Conjugates for treating diseases caused by psma expressing cells

ABSTRACT

The invention described herein pertains to the diagnosis, imaging, and/or treatment of pathogenic cell populations. In particular, the invention described herein pertains to the diagnosis, imaging, and/or treatment of diseases caused by PSMA expressing cells, such as prostate cancer cells, using compounds capable of targeting PSMA expressing cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 61/726,991, filed Nov. 15, 2012. U.S.Provisional Application Ser. No. 61/788,382, filed Mar. 15, 2013, andU.S. Provisional Application Ser. No. 61/875,971, filed Sep. 10, 2013,in which all of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The invention described herein pertains to the diagnosis, imaging,and/or treatment of pathogenic cell populations. In particular, theinvention described herein pertains to the diagnosis, imaging, and/ortreatment of diseases caused by PSMA expressing cells, such as prostatecancer cells, using compounds capable of targeting PSMA expressingcells.

BACKGROUND AND SUMMARY OF THE INVENTION

The prostate is a male reproductive organ and functions to produce andstore seminal fluid that provides nutrients and fluids for the survivalof sperm introduced into the vagina during reproduction. Like othertissues, the prostate gland may develop either malignant (cancerous) orbenign (non-cancerous) tumors. In fact, prostate cancer is one of themost common male cancers in western societies, and is the second leadingform of malignancy among American men. Current treatment methods forprostate cancer include hormonal therapy, radiation therapy, surgery,chemotherapy, photodynamic therapy, and combination therapy. However,many of these treatments affect the quality of life of the patient,especially for those men who are diagnosed with prostate cancer over age50. For example, the use of hormonal drugs is often accompanied by sideeffects such as osteoporosis and liver damage. Such side effects mightbe mitigated by the use of treatments that are more selective orspecific to the tissue being responsible for the disease state, andavoid non-target tissues like the bones or the liver.

Prostate-specific membrane antigen (PSMA) is a biomarker that isoverexpressed on prostate cancer. PSMA is over-expressed in themalignant prostate tissues when compared to other organs in the humanbody such as kidney, proximal small intestine, and salivary glands. PSMAis also expressed on the neovasculature within many non-prostate solidtumors, including lung, colon, breast, renal, liver and pancreaticcarcinomas, but not on normal vasculature. PSMA is also expressedminimally in brain. PSMA is a type II cell surface membrane-boundglycoprotein with ˜110 kD molecular weight, including an intracellularsegment (amino acids 1-18), a transmembrane domain (amino acids 19-43),and an extensive extracellular domain (amino acids 44-750). While thefunctions of the intracellular segment and the transmembrane domains arecurrently believed to be insignificant, the extracellular domain isinvolved in several distinct activities. For example, PSMA plays a rolein the central nervous system, where it metabolizes N-acetyl-aspartylglutamate (NAAG) into glutamic and N-acetyl aspartic acid. PSMA alsoplays a role in the proximal small intestine where it removes γ-linkedglutamate from poly-γ-glutamated folate and α-linked glutamate frompeptides and small molecules. However, PSMA's particular function onprostate cancer cells remains unresolved.

Unlike many other membrane-bound proteins, PSMA undergoes rapidinternalization into the cell in a similar fashion to cell surface boundreceptors like vitamin receptors. PSMA is internalized throughclathrin-coated pits and subsequently can either recycle to the cellsurface or go to lysosomes. Accordingly, diagnostic, imaging, andtherapeutic agents can be targeted to PSMA for delivery into PSMAexpressing cells, such as prostate cancer cells.

Described herein are compounds capable of binding to PSMA. Alsodescribed herein are compounds capable of targeting PSMA for delivery ofdiagnostic, imaging, and therapeutic agents. Also described herein arecompounds and compositions, and methods and uses thereof for diagnosing,imaging, and treating diseases caused by pathogenic populations of cellsthat express, or overexpress, PSMA.

It has been unexpectedly discovered that the conjugates described hereinexhibit high affinity for PSMA. It has also been discovered that thecompounds described herein are efficacious in treating diseases causedby pathogenic cells that express PSMA, such a prostate cancer cells.

In one illustrative embodiment of the invention, PSMA binding drugdelivery conjugates of the formula

B-L-(D)_(n)

or pharmaceutically acceptable salts thereof are described herein, whereB comprises a urea or thiourea of lysine and an amino acid, or one ormore carboxylic acid derivatives thereof, where the urea or thiourea iscapable of binding to PSMA, L is a polyvalent linker, D is a radical ofa drug, and n is an integer selected from 1, 2, 3, and 4. It is to beunderstood that as used herein, such drugs, and the term drug, includestherapeutic agents, diagnostic agents, imaging agents, and othercompounds that are desirably delivered to or targeted to PSMA and/orPSMA expressing cells.

In another illustrative embodiment, PSMA binding drug deliveryconjugates of the formula

B-L-(D)_(n)

or pharmaceutically acceptable salts thereof are described herein, whereB is a radical of a PSMA binding or targeting ligand, L is a polyvalentlinker comprising an aminomethylphenylacetic acid diradical, or anaminophenylacetic acid diradical, or both, D is a radical of a drug, andn is an integer selected from 1, 2, 3, and 4.

It is to be understood that every combination of the various embodimentsof each of B, L, D, and n described herein form illustrative embodimentsof the conjugates of the invention, whether those various embodiments ofeach of B, L. D are species, subgenera, or genera. It is to be furtherunderstood that each of those additional illustrative embodiments ofcompounds may be used in any of the compositions, unit doses, methods,and/or uses described herein.

In another embodiment, pharmaceutical compositions containing one ormore of the compounds are also described herein. In one aspect, thecompositions are in bulk form and are suitable for preparing unit doses,unit dosage forms, and the like that may be included in the uses and/ormethods described herein. In another aspect, the compositions include atherapeutically effective amount of the one or more compounds fordiagnosis, imaging, and/or treatment of diseases caused by PSMAexpressing cells in a patient. Illustrative compositions include unitdoses, unit dosage forms, and the like. It is to be understood that thecompositions may include other components and/or ingredients, including,but not limited to, other therapeutically active compounds, and/or oneor more carriers, and/or one or more diluents, and/or one or moreexcipients, and the like. In another embodiment, methods for using thecompounds and pharmaceutical compositions for diagnosis, imaging, and/ortreatment of diseases caused by PSMA expressing cells in a patient arealso described herein. In one aspect, the methods include the step ofadministering one or more of the compounds and/or compositions describedherein to the patient. In another embodiment, uses of the compounds andcompositions in the manufacture of a medicament for diagnosis, imaging,and/or treatment of diseases caused by PSMA expressing cells in apatient are also described herein. In one aspect, the medicamentsinclude a therapeutically effective amount of the one or more compoundsand/or compositions described herein.

It is appreciated herein that the compounds described herein may be usedalone or in combination with other compounds useful for diagnosis,imaging, and/or treatment of diseases caused by PSMA expressing cells ina patient, including those compounds that may be therapeuticallyeffective by the same or different modes of action. In addition, it isappreciated herein that the compounds described herein may be used incombination with other compounds that are administered to treat othersymptoms of the disease, such as compounds administered to decreasepain, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative affinity of (▪) PMPA, 1.0 (normalized); (●)DUPA, 0.05 (19-fold lower); (∘) EC1067, 30×; (□) EC1069, 22×; and (▾)EC1080, 6× in 10% serum/FDRPMI for PSMA.

FIG. 2 shows the relative affinity of (▪) PMPA, 1.0 (normalized); (●)EC1100, 20×; (▾) EC1168, 17×; (▴) EC1169, 7×; and (□) EC1170, 7× in 10%serum/FDRPMI for PSMA.

FIG. 3 shows the dose response and IC50 for EC1169 against LNCaP cells(2 h-72 h) as determined by ³H-thymidine incorporation cells in vitro.

FIG. 4 shows the dose response and IC50 for (▾) EC1718, (♦) EC1677, (▴)EC1719, (●) EC1720, and (▪) EC1721 against LNCaP cells (2 h-72 h) asdetermined by ³H-thymidine incorporation cells in vitro.

FIG. 5 shows the in vivo efficacy of EC1169 (c), EC1550 (●), and EC1551(▪), each at 2 μmol/kg, TIW (three times per week), 2 weeks, comparedagainst vehicle-treated controls (♦) in treating LNCaP tumor xenographs.

FIG. 6 shows that EC169 (c), EC1550 (●), and EC1551 (▪), each at 2μmol/kg, TIW, 2 weeks, compared against vehicle-treated controls (♦) donot exhibit gross animal toxicity.

FIG. 7 shows the in vivo efficacy of EC1584 (▾) and EC1588 (▴) each at 2μmol/kg, TIW, 2 weeks, compared against vehicle-treated controls (●) intreating LNCaP tumor xenographs.

FIG. 8 shows that EC1584 (▾) and EC1588 (▴), each at 2 μmol/kg, TIW, 2weeks, compared against vehicle-treated controls (●) do not exhibitgross animal toxicity.

FIG. 9 shows the in vivo efficacy of EC1169 (●) at 2 μmol/kg, TIW, 2weeks, compared to docetaxel, at 10 mg/kg, BIW, 2 weeks, MTD (▾), andeach compared to vehicle-treated control (▪) in treating LNCaP tumorxenographs.

FIG. 10 shows that of EC1169 (●) administered at 2 μmol/kg, TIW, 2weeks, exhibits substantially less gross animal toxicity compared todocetaxel, administered at 10 mg/kg, BIW, 2 weeks, MTD (▾).

FIG. 11 shows the in vivo efficacy of (▪) EC1718; (▴) EC1720; (▾)EC1721; (♦) EC1719; and (◯) EC1677, each administered at 2 μmol/kg, TIW,2 weeks; compared to (●) vehicle-treated control in treating LNCaP tumorxenographs.

FIG. 12 shows that (▪) EC1718; (▴) EC1720; (▾) EC1721; (♦) EC1719; and(◯) EC1677; compared to (●) vehicle-treated control, do not exhibitgross animal toxicity.

DETAILED DESCRIPTION

Several illustrative embodiments of the invention are described by thefollowing enumerated clauses:

1. A conjugate of the formula

B-L-(D)_(n)

or a pharmaceutically acceptable salt thereof, wherein B comprises aurea or thiourea of lysine and an amino acid, or one or more carboxylicacid derivatives thereof, including, but not limited to ureas orthioureas of lysine and aspartic acid, or glutamic acid, or homoglutamicacid, where the urea or thiourea is capable of binding to PSMA, L is apolyvalent linker, D is a radical of a drug, and n is an integerselected from 1, 2, 3, and 4.

2. A conjugate of the formula

B-L-(D)_(n)

or a pharmaceutically acceptable salt thereof, wherein B is a radical ofthe formula

L is a polyvalent linker, D is a radical of a drug, and n is an integerselected from 1, 2, 3, and 4.

3. The conjugate of clause 1 or 2 wherein L is a polyvalent linkercomprising an aminomethylphenylacetic acid diradical, or anaminophenylacetic acid diradical, or both.

4. A conjugate of the formula

B-L-(D)_(n)

or a pharmaceutically acceptable salt thereof, wherein B is a radical ofa PSMA binding ligand, L is a polyvalent linker comprising anaminomethylphenylacetic acid diradical or an aminophenylacetic aciddiradical or both, D is a radical of a drug, and n is an integerselected from 1, 2, 3, and 4.

5. The conjugate of clause 3 wherein B comprises a urea or thiourea oflysine and an amino acid, or one or more carboxylic acid derivativesthereof, including, but not limited to ureas or thioureas of lysine andaspartic acid, or glutamic acid, or homoglutamic acid.

6. The conjugate of any one of clauses 1 to 5 wherein B comprises a ureaor thiourea of lysine and glutamate, or one or more carboxylic acidderivatives thereof.

7. The conjugate of any one of clauses 1 to 5 wherein B comprises a ureaof lysine and glutamate.

8. The conjugate of any one of clauses 1 to 5 wherein B comprises a ureaor thiourea of L-lysine and L-glutamate, or one or more carboxylic acidderivatives thereof.

9. The conjugate of any one of clauses 1 to 5 wherein B comprises a ureaof L-lysine and L-glutamate.

10. The conjugate of any one of clauses 1 to 5 wherein B comprises aurea or thiourea of lysine and glutamic acid.

11. The conjugate of any one of clauses 1 to 5 wherein B comprises aurea or thiourea of D-lysine and D-glutamic acid.

12. The conjugate of any one of clauses 1 to 5 wherein B comprises aurea or thiourea of D-lysine and one or the following:

13. The conjugate of any one of clauses 1 to 5 wherein B comprises aurea or thiourea of D-lysine and:

14. The conjugate of any one of clauses 1 to 5 wherein B is a urea.

15. The conjugate of any one of clauses 1 to 5 wherein B is selectedfrom the following

16. The conjugate of any one of clauses 1 to 5 wherein B is selectedfrom the following

17. The conjugate of any one of clauses 1 to 5 wherein B is of theformula

The conjugate of any one of the preceding clauses wherein n is 1, 2, or3.

The conjugate of any one of the preceding clauses wherein n is 1 or 2.

The conjugate of any one of the preceding clauses wherein n is 1.

The conjugate of any one of the preceding clauses wherein at least onedrug is an imaging agent.

The conjugate of any one of the preceding clauses wherein at least onedrug is a diagnostic agent.

The conjugate of any one of the preceding clauses wherein at least onedrug is a therapeutic agent.

The conjugate of any one of the preceding clauses wherein at least onedrug is a cytotoxic agent.

The conjugate of any one of the preceding clauses wherein at least onedrug is a tubulysin.

The conjugate of any one of the preceding clauses wherein at least onedrug is a naturally occurring tubulysin.

The conjugate of any one of the preceding clauses wherein at least onedrug is tubulysin B.

The conjugate of any one of the preceding clauses wherein at least onedrug is a tubulysin of the formula

and pharmaceutical salts thereof are described, where

n is 1-3:

V is hydrogen, OR², or halo, and W is hydrogen, OR², or alkyl, where R²is independently selected in each instance from hydrogen, alkyl, andC(O)R³, where R³ is alkyl, cycloalkyl, alkenyl, aryl, or arylalkyl, eachof which is optionally substituted; providing that R² is not H when bothV and W are OR²; or V and W are taken together with the attached carbonto form a carbonyl;

X is hydrogen, alkyl, such as C₁₋₆ alkyl, or C₂₋₆ alkyl, C₁₋₄ alkyl, orC₂₋₄ alkyl, or alkenyl, such as C₂₋₆ alkenyl or C₂₋₄ alkenyl, each ofwhich is optionally substituted;

Z is alkyl or C(O)R⁴, where R⁴ is alkyl, CF₃, or aryl;

Ar is aryl or heteroaryl, each of which is optionally substituted; and

R is OH or R and the carbonyl to which it is attached is a carboxylicacid derivative.

The conjugate of any one of the preceding clauses wherein Ar isoptionally substituted phenyl.

The conjugate of any one of the preceding clauses wherein Ar is phenylsubstituted with one or more substituents selected from the groupconsisting of halo, hydroxy, amino, thio, carboxylate or a derivativethereof, sulfinyl or a derivative thereof, sulfonyl or a derivativethereof, phosphinyl or a derivative thereof, or phosphonyl or aderivative thereof, or alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteroalkyl,cycloheteroalkenyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl,each of which is optionally substituted.

The conjugate of any one of the preceding clauses wherein Ar is phenyl.

The conjugate of any one of the preceding clauses wherein Ar is4-hydroxyphenyl.

The conjugate of any one of the preceding clauses wherein X is CH₂QR⁹,where Q is —N—, —O—, or —S—; R⁹ is hydrogen or alkyl, alkenyl,cycloalkyl, aryl, or arylalkyl, each of which is optionally substituted,or C(O)R¹⁰.

The conjugate of any one of the preceding clauses wherein Q is O.

The conjugate of any one of the preceding clauses wherein R⁹ isoptionally substituted alkyl.

The conjugate of any one of the preceding clauses wherein R⁹ is alkyl.

The conjugate of any one of the preceding clauses wherein R¹⁰ isoptionally substituted alkyl.

The conjugate of any one of the preceding clauses wherein R¹⁰ is alkyl.

The conjugate of any one of the preceding clauses wherein at least onedrug is selected from the following:

The conjugate of any one of the preceding clauses wherein at least onedrug is:

The conjugate of any one of the preceding clauses wherein at least one Dis a radical of the formula

The conjugate of any one of the preceding clauses wherein at least one Dis a radical of the formula

The conjugate of any one of the preceding clauses wherein at least one Dis a radical of the formula

The conjugate of any one of the preceding clauses wherein at least one Dis a radical of the formula

The conjugate of any one of the preceding clauses wherein at least one Dis a radical of the formula

where n=1, 2, 3, 4, 5, or 6.

The conjugate of any one of the preceding clauses wherein L comprises anaminomethylphenylacetic acid diradical.

The conjugate of any one of the preceding clauses wherein L comprises anaminophenylacetic acid diradical

The conjugate of any one of the preceding clauses wherein L forms a ureaor thiourea with the lysine.

The conjugate of any one of the preceding clauses wherein L forms a ureawith the lysine.

The conjugate of any one of the preceding clauses wherein L forms anamide or thioamide with the lysine.

The conjugate of any one of the preceding clauses wherein L forms anamide with the lysine.

The conjugate of any one of the preceding clauses wherein L comprisesone or more aspartic acid diradicals.

The conjugate of any one of the preceding clauses wherein L comprisestwo or more aspartic acid diradicals.

The conjugate of the preceding clauses wherein the aspartic aciddiradicals are L-aspartic acid diradicals.

The conjugate of any one of the preceding clauses wherein L comprises acysteine diradical.

The conjugate of any one of the preceding clauses wherein L comprises aL-cysteine diradical.

The conjugate of any one of the preceding clauses wherein L comprisesL-Asp-L-Asp-L-Cys.

The conjugate of any one of the preceding clauses wherein L is areleasable linker, such as a releasable linker that is cleaved underconditions encountered at or near, or inside of pathogenic cellsexpressing, preferentially expressing, or overexpressing PSMA.

The conjugate of any one of the preceding clauses wherein L comprises adisulfide.

The conjugate of any one of the preceding clauses wherein L comprises acysteine disulfide diradical.

The conjugate of any one of the preceding clauses wherein L comprises aL-cysteine disulfide diradical.

The conjugate of any one of the preceding clauses wherein L comprisesL-Asp-L-Asp-L-Cys(S—S).

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula O—C(O)—N.

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula O—C(O)—NH.

The conjugate of any one of the preceding clauses wherein L and at leastone D taken together comprise a diradical of the formula O—C(O)—N.

The conjugate of any one of the preceding clauses wherein L and at leastone D taken together comprise a diradical of the formula O—C(O)—NH.

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula S—(CH₂)_(m)—O, where m is 2, 3, or 4.

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula S—(CH₂)_(m)—O—C(O)—N, where m is 2, 3, or 4.

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula S—(CH₂)_(m)—O—C(O)—NH, where m is 2, 3, or 4.

The conjugate of any one of the preceding clauses wherein L and at leastone D taken together comprise a diradical of the formulaS—(CH₂)_(m)—O—C(O)—N, where m is 2, 3, or 4.

The conjugate of any one of the preceding clauses wherein L and at leastone D taken together comprise a diradical of the formulaS—(CH₂)_(m)—O—C(O)—NH, where m is 2, 3, or 4.

The conjugate of any one of the preceding clauses wherein the terminalsulfur atom forms a disulfide.

The conjugate of any one of the preceding clauses wherein m is 2.

The conjugate of any one of the preceding clauses wherein L comprises achain of at least about 7 atoms, at least about 8 atoms, at least about9 atoms, at least about 10 atoms, at least about 11 atoms, at leastabout 12 atoms, at least about 13 atoms, at least about 14 atoms, or atleast about 15 atoms.

The conjugate of any one of the preceding clauses wherein L comprises achain of at least about 16 atoms, at least about 17 atoms, at leastabout 18 atoms, at least about 19 atoms, at least about 20 atoms, atleast about 21 atoms, at least about 22 atoms, at least about 23 atoms,at least about 24 atoms, at least about 25 atoms, or at least about 26atoms.

The conjugate of any one of the preceding clauses wherein L comprises achain of between about 7 and about 35 atoms, between about 7 and about30 atoms, or between about 7 and about 26 atoms.

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses where L comprises adiradical of the formula

The conjugate of any one of the preceding clauses where L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses where L comprises adiradical of the formula

The conjugate of any one of the preceding clauses where L comprises adiradical of the formula

The conjugate of any one of the preceding clauses where L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein L comprises adiradical of the formula

The conjugate of any one of the preceding clauses wherein B-L comprisesa diradical of the formula

The conjugate of any one of the preceding clauses wherein B-L comprisesa diradical of the formula

The conjugate of any one of the preceding clauses wherein B-L comprisesa diradical of the formula

The conjugate of any one of the preceding clauses wherein B-L comprisesa diradical of the formula

A conjugate of the formula

or a pharmaceutically acceptable salt thereof, and/or a hydrate, and/ora solvate, and/or a co-crystal of the foregoing; where D is radical of adrug.

A conjugate of the formula

or a pharmaceutically acceptable salt thereof, and/or a hydrate, and/ora solvate, and/or a co-crystal of the foregoing; where D is radical of adrug.

A conjugate the formula

or a pharmaceutically acceptable salt thereof, and/or a hydrate, and/ora solvate, and/or a co-crystal of the foregoing; where D is radical of adrug.

A conjugate of the formula

or a pharmaceutically acceptable salt thereof, and/or a hydrate, and/ora solvate, and/or a co-crystal of the foregoing; where D is radical of adrug.

A pharmaceutical composition comprising one or more of the compounds orconjugates of any one of the preceding clauses.

A pharmaceutical composition comprising one or more of the compounds orconjugates of any one of the preceding clauses for treating a disease ina host animal caused by a pathogenic population of cells, said cellsexpressing PSMA.

A unit dose or unit dosage form in single or divided form, the unit doseor unit dosage form comprising a therapeutically effective amount of oneor more of the compounds or conjugates of any one of the precedingclauses for treating a disease in a host animal caused by a pathogenicpopulation of cells, said cells expressing PSMA.

The composition or unit dose or unit dosage form of any one of thepreceding clauses further comprising one or more carriers, diluents, orexcipients, or a combination thereof.

A method for treating a disease in a host animal caused by a pathogenicpopulation of cells, said cells expressing PSMA, the method comprisingthe step of administering to the patient a composition comprising atherapeutically effective amount of one or more of the compounds orconjugates or one or more of the compositions or unit doses or unitdosage forms of any one of clauses 1 to 73

Use of one or more of the compounds or conjugates, compositions, unitdoses, or unit dosage forms of any one of the preceding clauses in themanufacture of a medicament for treating a disease in a host animalcaused by a pathogenic population of cells, said cells expressing PSMA.

The composition, unit doses or unit dosage form, method, or use of anyone of the preceding clauses wherein the cells are prostate cancercells.

The composition, unit doses or unit dosage form, method, or use of anyone of the preceding clauses wherein the disease is prostate cancer.

The composition, unit doses or unit dosage form, method, or use of anyone of the preceding clauses wherein the host animal is a human.

In reciting the foregoing and following collection of embodiments andclauses, it is to be understood that all possible combinations offeatures, and all possible subgenera and sub-combinations are described.For example, it is to be understood that when B is limited to a bindingligand comprising urea of L-lysine and L-glutamate, L may be limited toa linker comprising one or more aspartic acid diradicals, oralternatively, to comprising a cysteine diradical, or alternatively,comprising L-Asp-L-Asp-L-Cys(S—S), and so forth. Similarly, when D islimited to a naturally occurring tubulsyin, L may be limited to a linkercomprising diradical of the formula S—(CH₂)_(m)—O—C(O)—N, oralternatively, to comprising a cysteine disulfide diradical, oralternatively, comprising an aminophenylacetic acid diradical, and soforth. Similarly, when B is limited to a binding ligand comprising aurea or thiourea of lysine and glutamate, or one or more carboxylic acidderivatives thereof. L may be limited to a linker comprising one or moreD-aspartic acid diradicals, and D may be limited to a tubulysin, oralternatively, L may be limited to a linker comprising a diradical ofthe formula O—C(O)—N, and D may be limited to an imaging agent, oralternatively, L may be limited to a linker comprising a diradical ofthe formula S—(CH₂)_(m)—O—C(O)—NH, and D may be limited to a therapeuticagent, and so forth. Other combinations, subgenera and sub-combinationsare also described by the collection of clauses.

In another embodiment, at least one drug is an imaging agent.Illustrative imaging agents for the conjugates described herein include,but are not limited to, radioisotopes, such as a radioactive isotope ofa metal coordinated to a chelating group. Illustrative radioactive metalisotopes include technetium, rhenium, gallium, gadolinium, indium,copper, and the like, including isotopes ¹¹¹In, ^(99m)Tc, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, and the like. Additional illustrative examples ofradionuclide imaging agents are described in U.S. Pat. No. 7,128,893,the disclosure of which is incorporated herein by reference. Additionalillustrative chelating groups are tripeptide or tetrapeptides, includingbut not limited to tripeptides having the formula:

wherein R is independently selected in each instance H, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which isoptionally substituted. It is to be understood that one R includes aheteroatom, such as nitro, oxygen, or sulfur, and is the point ofattachment of linker L. Illustratively, the following chelating groupsare described:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L, and n is an integer from 1 to about 5.

Illustrative imaging agents also include, but are not limited to,fluorescent agents, such as Oregon Green fluorescent agents, includingbut not limited to Oregon Green 488, Oregon Green 514, and the like,AlexaFluor fluorescent agents, including but not limited to AlexaFluor488, AlexaFluor 647, and the like, fluorescein, and related analogs,BODIPY fluorescent agents, including but not limited to BODIPY F1,BODIPY 505, and the like, rhodamine fluorescent agents, including butnot limited to tetramethylrhodamine, and the like, DyLight fluorescentagents, including but not limited to DyLight 680, DyLight 800, and thelike, CW 800, IRdye 800CW, Texas Red, phycoerythrin, and others. Furtherillustrative fluorescent agents include compounds of the followingformula:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; Y is OR^(a), NR^(a) ₂, or NR^(a) ₃ ⁺; and Y′ is O, NR^(a), orNR^(a) ₂ ⁺; where each R is independently selected in each instance fromH, fluoro, sulfonic acid, sulfonate, and salts thereof, and the like;and R^(a) is hydrogen or alkyl. Further illustrative fluorescent agentsinclude compounds of the following formula:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; and each R is independently selected in each instance from H,alkyl, heteroalkyl, and the like; and n is an integer from 0 to about 4.

Illustrative imaging agents also include, but are not limited to, PETimaging agents, and FRET imaging agents. Illustrative PET imaging agentsinclude ¹⁸F, ¹¹C, ⁶⁴Cu, ⁶⁵Cu, and the like. Illustrative FRET imagingagents include ⁶⁴Cu, ⁶⁵Cu, and the like. It is to be understood that inthe case of ¹⁸F and ¹¹C, the imaging isotope may be directly attached tothe linker, or alternatively may be present on a structure attached tothe linker. For example in the case of ¹⁸F, fluoroaryl groups, such asfluorophenyl, difluorophenyl, fluoronitrophenyl, and the like aredescribed. For example in the case of ¹¹C, alkyl and alkyl aryl aredescribed.

In another embodiment, the drug can be any molecule capable ofmodulating or otherwise modifying cell function, includingpharmaceutically active compounds. Illustrative drugs include, but arenot limited to, peptides, oligopeptides, retro-inverso oligopeptides,proteins, protein analogs in which at least one non-peptide linkagereplaces a peptide linkage, apoproteins, glycoproteins, enzymes,coenzymes, enzyme inhibitors, amino acids and their derivatives,receptors and other membrane proteins; antigens and antibodies thereto;haptens and antibodies thereto; hormones, lipids, phospholipids,liposomes; toxins; antibiotics; analgesics; bronchodilators;beta-blockers; antimicrobial agents; antihypertensive agents;cardiovascular agents including antiarrhythmics, cardiac glycosides,antianginals and vasodilators; central nervous system agents includingstimulants, psychotropics, antimanics, and depressants; antiviralagents; antihistamines; cancer drugs including chemotherapeutic agents;tranquilizers; anti-depressants; H-2 antagonists; anticonvulsants;antinauseants; prostaglandins and prostaglandin analogs; musclerelaxants; anti-inflammatory substances; immunosuppressants, stimulants;decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics;anti-Parkinson agents; expectorants; cough suppressants; mucolytics; andmineral and nutritional additives.

Illustrative chemotherapeutic agents also include, but are not limitedto, compounds that are cytotoxic, enhance tumor permeability, inhibittumor cell proliferation, promote apoptosis, decrease anti-apoptoticactivity in target cells, used to treat diseases caused by infectiousagents, enhance an endogenous immune response directed to the pathogeniccells, or are useful for treating a disease state caused by thepathogenic cells. Such chemotherapeutic agents may operate by any of alarge variety of mechanisms of action. For example, cytotoxic compoundsmay disrupt any of a wide variety of cellular mechanisms that areimportant for cell survival and/or cell proliferation and/or cause celldeath or apoptosis.

Illustrative chemotherapeutic agents also include, but are not limitedto, adrenocorticoids and corticosteroids, alkylating agents,antiandrogens, antiestrogens, androgens, aclamycin and aclamycinderivatives, estrogens, antimetabolites such as cytosine arabinoside,purine analogs, pyrimidine analogs, and methotrexate, busulfan,carboplatin, chlorambucil, cisplatin and other platinum compounds,tamoxiphen, taxol, paclitaxel, paclitaxel derivatives. Taxotere®,cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids,prednisone, hydroxyurea, teniposide, mitomycins, discodermolides,microtubule inhibitors, epothilones, tubulysins, cyclopropylbenz[e]indolone, seco-cyclopropyl benz[e]indolone, O—Ac-seco-cyclopropylbenz[e]indolone, bleomycin and any other antibiotic, nitrogen mustards,nitrosureas, vinca alkaloids, such as vincristine, vinblastine,vindesine, vinorelbine and analogs and derivative thereof such asdeacetylvinblastine monohydrazide (DAVLBH), colchicine, colchicinederivatives, allocolchicine, thiocolchicine, trityl cysteine,halicondrin B, dolastatins such as dolastatin 10, amanitins such asα-amanitin, camptothecin, irinotecan, and other camptothecin derivativesthereof, geldanamycin and geldanamycin derivatives, estramustine,nocodazole, MAP4, colcemid, inflammatory and proinflammatory agents,peptide and peptidomimetic signal transduction inhibitors, rapamycins,such as sirolimus and everolimus, and any other drug or toxin.

In another embodiment, at least one drug is selected from cryptophycins,bortezomib, thiobortezomib, tubulysins, aminopterin, rapamycins, such aseverolimus and sirolimus, paclitaxel, docetaxel, doxorubicin,daunorubicin, α-amanatin, verucarin, didemnin B, geldanomycin,purvalanol A, ispinesib, budesonide, dasatinib, epothilones,maytansines, and tyrosine kinase inhibitors, including analogs andderivatives of each of the foregoing.

Other drugs that can be included in the conjugates described hereininclude amphotericin B, acyclovir, trifluridine, ganciclovir,zidovudine, amantadine, ribavirin, and the like.

In another embodiment, at least one drug is a tubulysin. As used herein,the term “tubulysin” generally refers to the compounds described hereinand analogs and derivatives thereof. It is also to be understood thatany corresponding pharmaceutically acceptable salt is also included inthe illustrative embodiments described herein. Illustrative derivativesof tubulysins include, but are not limited to, those compounds that maybe synthetically prepared from the compounds described herein. It is tobe understood that such derivatives may include prodrugs of thecompounds described herein, compounds described herein that include oneor more protection or protecting groups, including compounds that areused in the preparation of other compounds described herein.

As described herein, the tubulysin compounds may be inhibitors oftubulin polymerization, and also may be DNA-alkylators.

Illustrative tubulysins include, but are not limited to compounds of theformula

and pharmaceutical salts thereof are described, where

n is 1-3;

V is hydrogen, OR², or halo, and W is hydrogen, OR², or alkyl, where R²is independently selected in each instance from hydrogen, alkyl, andC(O)R³, where R³ is alkyl, cycloalkyl, alkenyl, aryl, or arylalkyl, eachof which is optionally substituted; providing that R² is not H when bothV and W are OR²; or V and W are taken together with the attached carbonto form a carbonyl:

X is hydrogen, alkyl, such as C₁₋₄ alkyl, or alkenyl, such as C₂₋₄alkenyl, each of which is optionally substituted;

Z is alkyl or C(O)R⁴, where R⁴ is alkyl, CF₃, or aryl; or when Y ispresent, Z is alkyl; and Y is O:

Ar is aryl, such as phenyl, or heteroaryl, each of which is optionallysubstituted; and

R is OH or R and the carbonyl to which it is attached is a carboxylicacid derivative, such as an acylhydrazide.

In another embodiment, X is CH₂QR⁹, where Q is —N—, —O—, or —S—; R⁹ ishydrogen or alkyl, alkenyl, cycloalkyl, aryl, or arylalkyl, each ofwhich is optionally substituted, or C(O)R¹⁰, where R¹⁰ is hydrogen oralkyl, alkenyl, cycloalkyl, aryl, or arylalkyl In another embodiment, R⁹and Q are taken together to form S(O)₂R¹⁰, P(O)(OR^(10a))₂, where R¹⁰and OR^(10a) are independently selected in each instance from the groupconsisting of hydrogen, and alkyl, alkenyl, cycloalkyl, aryl,heteroaryl, and arylalkyl, each of which is optionally substituted, orR^(10a) is a metal cation.

In another embodiment, X is H. Illustrative examples of such compounds,and their preparation are described in J. Med. Chem. 10.1021/jm701321p(2008), the disclosure of which is incorporated herein by reference.

In another embodiment, X is a radical of the formula

where R¹² represents 1 or more substituents selected from alkyl,alkenyl, cycloalkyl, aryl, and arylalkyl, each of which is optionallysubstituted. It is to be understood that other olefins may form byisomerization, depending on the conditions of the reaction and theidentity of R¹². For example, when R¹² is alkyl, it is appreciated thatunder the reaction conditions, the double bond can migrate to othercarbon atoms along the alkenyl chain, including to form the terminal orω-olefin.

In another embodiment, X is a radical of the formula

where R¹³ is C(O)R¹⁰, C(O)OR¹⁰ or CN, where R¹⁰ is independentlyselected in each instance.

In another embodiment. X is CH₂—OH.

In another embodiment, X is CH₂—X^(A), where X^(A) is halogen.OS(O)₂R¹⁰. OP(O)(OR^(10a))R¹⁰, or OP(O)(OR^(10a))₂; where R¹⁰ andR^(10a) are independently selected in each instance from the groupconsisting of hydrogen, alkyl, alkenyl, cycloalkyl, aryl, and arylalkyl,each of which is optionally substituted, or R^(10a) is a metal cation.

In another embodiment of any of the foregoing embodiments, Ar isoptionally substituted aryl. In another embodiment of any of theforegoing embodiments, Ar is a radical of the formula

where R¹ is hydrogen, or R¹ represents 1 to 3 substituents independentlyselected from the group consisting of halo, nitro, carboxylate or aderivative thereof, cyano, hydroxyl, alkyl, haloalkyl, alkoxy,haloalkoxy, and OR⁶, where R⁶ is hydrogen or optionally substitutedalkyl, heteroalkyl, aryl, a phenol protecting group, a prodrug moiety,C(O)R⁷, P(O)(OR⁸)₂, or SO₃R⁸, where R⁷ and R⁸ are independently selectedin each instance from hydrogen, or alkyl, alkenyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, and arylalkyl, each of which isoptionally substituted, or R⁸ is a metal cation are described.

In another embodiment of any of the foregoing embodiments, Z is methyl.In another embodiment of any of the foregoing embodiments, R¹ is H. Inanother embodiment of any of the foregoing embodiments, R¹ is OR⁶ atC(4), where R⁶ is hydrogen, alkyl, or COR⁷. In another embodiment of anyof the foregoing embodiments, V is hydrogen, and W is OC(O)R³. Inanother embodiment of any of the foregoing embodiments. V is hydrogen,and W is acetyloxy.

In another embodiment of any of the foregoing embodiments, the compoundsof the various formulae have the following absolute configuration:

at each of the indicated asymmetric carbon atoms.

Additional illustrative tubulysins that are useable in the conjugatesdescribed herein include the following:

Tubulysin X^(B) EC0313 —O—CH₃ EC0346 —O—(CH₂)₂—OH EC0356—O—(CH₂)₂CH(CH₃)₂ EC0374 —S—(CH₂)₂—SH EC0386 —OH EC0550 —(CH₂)₂—CH═CH₂EC0560 —S—(CH₂)₂—OH EC0575 —O—C(O)—(CH═CH)—CH₂—Cl EC0585—NH—C(O)—CH₂CH(CH₃)₂ EC0611 —O—(CH₂)₂CH₃ EC0623 —S—(CH₂)₂CH₃and pharmaceutical salts thereof.

In another embodiment, the tubulysin is a naturally occurring tubulysin.Natural tubulysins are generally linear tetrapeptides consisting ofN-methyl pipecolic acid (Mep), isoleucine (Ile), an unnatural aminoacidcalled tubuvalin (Tuv), and either an unnatural aminoacid calledtubutyrosine (Tut, an analog of tyrosine) or an unnatural aminoacidcalled tubuphenylalanine (Tup, an analog of phenylalanine). In anotherembodiment, naturally occurring tubulysins, and analogs and derivativesthereof, of the following general formula are described

and pharmaceutical salts thereof, where Ar, R, and R¹⁰ are as describedin the various embodiments herein.

In another embodiment, the naturally occurring tubulysins of thefollowing general formula are described

Factor R¹⁰ R¹ A (CH₃)₂CHCH₂ OH B CH₃(CH₂)₂ OH C CH₃CH₂ OH D (CH₃)₂CHCH₂H E CH₃(CH₂)₂ H F CH₂CH₃ H G (CH₃)₂C═CH OH H CH₃ H I CH₃ OHand pharmaceutical salts thereof.

It is to be understood that the conjugate of the tubulysin or analog orderivative thereof may be formed at any position. Illustratively,conjugates of tubulysins are described where the linker (L) is attachedto any of the following positions:

where the (*) symbol indicates optional attachment locations.

In another embodiment, compounds are described herein where theconjugate is formed at the terminal carboxylic acid group or theterminal acylhydrazine derivative group of each of the tubulysinsdescribed herein.

Additional tubulysins useful in preparing the conjugates describedherein are described in US patent application publication Nos.2006/0128754 and 2005/0239713, the disclosures of which are incorporatedherein by reference. Additional tubulysins useful in preparing theconjugates described herein are described in co-pending U.S. patentapplication publication No. 2010/0240701 the disclosure of which isincorporated herein by reference. Tubulysins may also be prepared aredescribed in Peltier et al., “The Total Synthesis of Tubulysin D,” J.Am. Chem. Soc. 128:16018-19 (2006), the disclosure of which isincorporated herein by reference.

In another embodiment, at least one drug is a rapamycin. As used herein,the term “a rapamycin” is understood to include sirolimus (rapamycin),temsirolimus, everolimus, and ridaforolimus, and related compounds, andcompounds of the formula

and pharmaceutically acceptable salts thereof, wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C):

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In another embodiment, at least one drug is a vinca alkaloids, such asvincristine, vinblastine, vindesine, vinorelbine and analogs andderivative thereof such as deacetylvinblastine monohydrazide (DAVLBH).

In another embodiment, at least one drug is a mitomycin, or an analog orderivative thereof.

In another embodiment, the conjugates described herein include at leasttwo drugs, including those described herein, In one variation, the drugsare the same. In another variation, at least two of the drugs aredifferent. In another variation, the two or more drugs are selected fromvinca alkaloids, cryptophycins, bortezomib, thiobortezomib, tubulysins,aminopterin, rapamycins, such as everolimus and sirolimus, paclitaxel,docetaxel, doxorubicin, daunorubicin, α-amanatin, verucarin, didemnin B,geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib,epothilones, maytansines, and tyrosine kinase inhibitors, includinganalogs and derivatives of each of the foregoing.

As used herein, the term “linker” includes is a chain of atoms thatconnects two or more functional parts of a molecule to form a conjugate.Illustratively, the chain of atoms is selected from C, N, O, S, Si, andP, or C, N, O, S, and P, or C, N, O, and S. The chain of atomscovalently connects different functional capabilities of the conjugate,such as binding ligands, drugs, diagnostic agents, imaging agents, andthe like. The linker may have a wide variety of lengths, such as in therange from about 2 to about 100 atoms in the contiguous backbone. Theatoms used in forming the linker may be combined in all chemicallyrelevant ways, such as chains of carbon atoms forming alkylene,alkenylene, and alkynylene groups, and the like; chains of carbon andoxygen atoms forming ethers, polyoxyalkylene groups, or when combinedwith carbonyl groups forming esters and carbonates, and the like; chainsof carbon and nitrogen atoms forming amines, imines, polyamines,hydrazines, hydrazones, or when combined with carbonyl groups formingamides, ureas, semicarbazides, carbazides, and the like; chains ofcarbon, nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines,or when combined with carbonyl groups forming urethanes, amino acids,acyloxylamines, hydroxamic acids, and the like; and many others. Inaddition, it is to be understood that the atoms forming the chain ineach of the foregoing illustrative embodiments may be either saturatedor unsaturated, thus forming single, double, or triple bonds, such thatfor example, alkanes, alkenes, alkynes, imines, and the like may beradicals that are included in the linker. In addition, it is to beunderstood that the atoms forming the linker may also be cyclized uponeach other or be part of cyclic structure to form divalent cyclicstructures that form the linker, including cyclo alkanes, cyclic ethers,cyclic amines, and other heterocycles, arylenes, heteroarylenes, and thelike in the linker. In this latter arrangement, it is to be understoodthat the linker length may be defined by any pathway through the one ormore cyclic structures. Illustratively, the linker length is defined bythe shortest pathway through the each one of the cyclic structures. Itis to be understood that the linkers may be optionally substituted atany one or more of the open valences along the chain of atoms, such asoptional substituents on any of the carbon, nitrogen, silicon, orphosphorus atoms. It is also to be understood that the linker mayconnect the two or more functional parts of a molecule to form aconjugate at any open valence, and it is not necessary that any of thetwo or more functional parts of a molecule forming the conjugate areattached at any apparent end of the linker.

In another embodiment, the linker (L) comprises a radical of the formula

where m1, m2, m3, n, p, q, and r are integers that are eachindependently selected from the range of 0 to about 8, providing that atleast one of m1, m2, m3, n, p, q, and r is not 0; AA is an amino acid;and drugs are optionally attached at one or more of the (*) atoms. It isto be understood that the drugs may be directly attached, or attachedthrough additional portions of the linker (L). In another embodiment, AAis a naturally occurring amino acid of either the natural or unnaturalconfiguration. In another embodiment, one or more of AA is a hydrophilicamino acid. In another embodiment, one or more of AA is Asp and/or Arg.In another embodiment, the integer n is 1 or greater. In anotherembodiment, the integer n is 2 or greater. In another embodiment, theinteger n is 3 or greater. In another embodiment, the integer n is 4 orgreater. In another embodiment, the integer n is 5 or greater. Inanother aspect, the integer q is 1 or greater. In another embodiment,the integer m1 is 1 or greater. In another embodiment, the integer m1is 1. In another embodiment, the integer m2 is 1 or greater. In anotherembodiment, the integer m2 is 1. In another embodiment, the integer m3is 1 or greater. In another embodiment, the integer m3 is 1. In anotherembodiment, the integer p is 1 or greater. In another embodiment, theinteger p is 1. In another embodiment, the integer p is 2. In anotherembodiment, the integer q is 1 or greater. In another embodiment, theinteger q is 1. In another embodiment, the integer q is 2. In anotherembodiment, the integer r is 1 or greater. In another embodiment, theinteger r is 1. In another embodiment, the integer r is 2.

It is to be understood that all combinations of the foregoingembodiments are described herein. For example, in another embodiment, nis 1 or greater, and m1 is one or greater, or n is 1 or greater, m1 is1, and q is 1; and so forth. For example, in another embodiment, n is 1or greater, and m2 is one or greater, or n is 2 or greater, m2 is 1, andq is 1; or n is 2 or greater, m3 is 1, q is 1, and p is 1; and so forth.For example, in another embodiment, n is 1 or greater, and m1 is one orgreater; or n is 2 or greater, m3 is 1, and q is 1; or n is 2 orgreater, m2 is 1, q is 1, and p is 1; or n is 2 or greater, m1 is 1, qis 1, and r is 1; or n is 2 or greater, m3 is 1, q is 1, p is 1, and ris 1; and so forth.

In another embodiment, the polyvalent linker includes one or moredivalent hydrophilic radicals, as described herein, which may also bereferred to as spacer linkers. It is appreciated that the arrangementand/or orientation of the various hydrophilic linkers may be in a linearor branched fashion, or both. For example, the hydrophilic linkers mayform the backbone of the linker forming the conjugate between the ligandand the one or more drugs. Alternatively, the hydrophilic portion of thelinker may be pendant to or attached to the backbone of the chain ofatoms connecting the binding ligand B to the one or more drugs D. Inthis latter arrangement, the hydrophilic portion may be proximal ordistal to the backbone chain of atoms.

In another embodiment, the linker is generally linear, and thehydrophilic groups are arranged generally in a series to form achain-like linker in the conjugate. Said another way, the hydrophilicgroups form some or all of the backbone of the linker in such a linearlinker embodiment.

In another embodiment, the linker is branched with hydrophilic groups.In this branched embodiment, the hydrophilic groups may be proximal tothe backbone or distal to the backbone. In each of these arrangements,the linker is generally more spherical or cylindrical in shape. Inanother embodiment, the linker is shaped like a bottle-brush. In anotherembodiment, the backbone of the linker is formed by a linear series ofamides, and the hydrophilic portion of the linker is formed by aparallel arrangement of branching side chains, such as by connectingmonosaccharides, sulfonates, and the like, and derivatives and analogsthereof.

It is understood that the linker (L) may be neutral or ionizable undercertain conditions, such as physiological conditions encountered invivo. For ionizable linkers, under the selected conditions, the linkermay deprotonate to form a negative ion, or alternatively becomeprotonated to form a positive ion. It is appreciated that more than onedeprotonation or protonation event may occur. In addition, it isunderstood that the same linker may deprotonate and protonate to forminner salts or zwitterionic compounds.

In another embodiment, the hydrophilic spacer linkers are neutral, an inparticular neutral under physiological conditions, the linkers do notsignificantly protonate nor deprotonate. In another embodiment, thehydrophilic spacer linkers may be protonated to carry one or morepositive charges. It is understood that the protonation capability iscondition dependent. In one aspect, the conditions are physiologicalconditions, and the linker is protonated in vivo. In another embodiment,the spacers include both regions that are neutral and regions that maybe protonated to carry one or more positive charges. In anotherembodiment, the spacers include both regions that may be deprotonated tocarry one or more negative charges and regions that may be protonated tocarry one or more positive charges. It is understood that in this latterembodiment that zwitterions or inner salts may be formed.

In another embodiment, the regions of the linkers that may bedeprotonated to carry a negative charge include carboxylic acids, suchas aspartic acid, glutamic acid, and longer chain carboxylic acidgroups, and sulfuric acid esters, such as alkyl esters of sulfuric acid.In another embodiment, the regions of the linkers that may be protonatedto carry a positive charge include amino groups, such aspolyaminoalkylenes including ethylene diamines, propylene diamines,butylene diamines and the like, and/or heterocycles includingpyrollidines, piperidines, piperazines, and other amino groups, each ofwhich is optionally substituted. In another embodiment, the regions ofthe linkers that are neutral include poly hydroxyl groups, such assugars, carbohydrates, saccharides, inositols, and the like, and/orpolyether groups, such as polyoxyalkylene groups includingpolyoxyethylene, polyoxypropylene, and the like.

In another embodiment, the hydrophilic spacer linkers described hereininclude are formed primarily from carbon, hydrogen, and oxygen, and havea carbon/oxygen ratio of about 3:1 or less, or of about 2:1 or less. Inanother embodiment, the hydrophilic linkers described herein include aplurality of ether functional groups. In another embodiment, thehydrophilic linkers described herein include a plurality of hydroxylfunctional groups. Illustrative fragments and radicals that may be usedto form such linkers include polyhydroxyl compounds such ascarbohydrates, polyether compounds such as polyethylene glycol units,and acid groups such as carboxyl and alkyl sulfuric acids. In onevariation, oligoamide spacers, and the like may also be included in thelinker.

Illustrative divalent hydrophilic linkers include carbohydrates such assaccharopeptides as described herein that include both a peptide featureand sugar feature; glucuronides, which may be incorporated via [2+3]Huisgen cyclization, also known as click chemistry; β-alkyl glycosides,such as of 2-deoxyhexapyranoses (2-deoxyglucose, 2-deoxyglucuronide, andthe like), and β-alkyl mannopyranosides. Illustrative PEG groups includethose of a specific length range from about 4 to about 20 PEG groups.Illustrative alkyl sulfuric acid esters may also be introduced withclick chemistry directly into the backbone. Illustrative oligoamidespacers include EDTA and DTPA spacers, β-amino acids, and the like.

In another embodiment, the polyvalent linker L comprises one or morepolyethers, such as the linkers of the following formulae:

where m is an integer independently selected in each instance from 1 toabout 8; p is an integer selected 1 to about 10; and n is an integerindependently selected in each instance from 1 to about 3. In oneaspect, m is independently in each instance 1 to about 3. In anotheraspect, n is 1 in each instance. In another aspect, p is independentlyin each instance about 4 to about 6. Illustratively, the correspondingpolypropylene polyethers corresponding to the foregoing are contemplatedherein and may be included in the conjugates as hydrophilic spacerlinkers. In addition, it is appreciated that mixed polyethylene andpolypropylene polyethers may be included in the conjugates ashydrophilic spacer linkers. Further, cyclic variations of the foregoingpolyether compounds, such as those that include tetrahydrofuranyl,1,3-dioxanes, 1,4-dioxanes, and the like are contemplated herein.

In another embodiment, the polyvalent linker L comprises a plurality ofhydroxyl functional groups, such as linkers that incorporatemonosaccharides, oligosaccharides, polysaccharides, and the like. It isto be understood that the polyhydroxyl containing spacer linkerscomprises a plurality of —(CROH)— groups, where R is hydrogen or alkyl.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer from 1to about 3; n is an integer from 1 to about 5, or from 2 to about 5, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one aspect, the integer n is 3 or 4. In another aspect, theinteger p is 3 or 4. In another aspect, the integer r is 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer from 1to about 3; n is an integer from 1 to about 5, or from 2 to about 5, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one aspect, the integer n is 3 or 4. In another aspect, theinteger p is 3 or 4. In another aspect, the integer r is 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following cyclic polyhydroxyl groups:

wherein n is an integer from 2 to about 5, p is an integer from 1 toabout 5, and r is an integer from 1 to about 4. In one aspect, theinteger n is 3 or 4. In another aspect, the integer p is 3 or 4. Inanother aspect, the integer r is 2 or 3. It is understood that allstereochemical forms of such sections of the linkers are contemplatedherein. For example, in the above formula, the section may be derivedfrom ribose, xylose, glucose, mannose, galactose, or other sugar andretain the stereochemical arrangements of pendant hydroxyl and alkylgroups present on those molecules. In addition, it is to be understoodthat in the foregoing formulae, various deoxy compounds are alsocontemplated. Illustratively, compounds of the following formulae arecontemplated:

wherein n is equal to or less than r, such as when r is 2 or 3, n is 1or 2, or 1, 2, or 3, respectively.

In another embodiment, the polyvalent linker L comprises one or morepolyhydroxyl radicals of the following formula:

wherein n and r are each an integer selected from 1 to about 3. In oneaspect, the linker includes one or more polyhydroxyl compounds of thefollowing formulae:

It is understood that all stereochemical forms of such sections of thelinkers are contemplated herein. For example, in the above formula, thesection may be derived from ribose, xylose, glucose, mannose, galactose,or other sugar and retain the stereochemical arrangements of pendanthydroxyl and alkyl groups present on those molecules.

In another embodiment, the polyvalent linker L comprises one or morepolyhydroxyl groups that are spaced away from the backbone of thelinker. In one embodiment, such carbohydrate groups or polyhydroxylgroups are connected to the back bone by a triazole group, formingtriazole-linked hydrophilic spacer linkers. Illustratively, the linkerincludes fragments of the following formulae:

wherein n, m, and r are integers and are each independently selected ineach instance from 1 to about 5. In one illustrative aspect, m isindependently 2 or 3 in each instance. In another aspect, r is 1 in eachinstance. In another aspect, n is 1 in each instance. In one variation,the group connecting the polyhydroxyl group to the backbone of thelinker is a different heteroaryl group, including but not limited to,pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazole, thienyl,thiazole, isothiazole, oxadiazole, and the like. Similarly, divalent6-membered ring heteroaryl groups are contemplated. Other variations ofthe foregoing illustrative hydrophilic spacer linkers includeoxyalkylene groups, such as the following formulae:

wherein n and r are integers and are each independently selected in eachinstance from 1 to about 5; and p is an integer selected from 1 to about4.

In another embodiment, the polyvalent linker L comprises one or morecarbohydrate groups or polyhydroxyl groups connected to the back bone byan amide group, forming amide-linked hydrophilic spacer linkers.Illustratively, such linkers include fragments of the followingformulae:

wherein n is an integer selected from 1 to about 3, and m is an integerselected from 1 to about 22. In one illustrative aspect, n is 1 or 2. Inanother illustrative aspect, m is selected from about 6 to about 10,illustratively 8. In one variation, the group connecting thepolyhydroxyl group to the backbone of the linker is a differentfunctional group, including but not limited to, esters, ureas,carbamates, acylhydrazones, and the like. Similarly, cyclic variationsare contemplated. Other variations of the foregoing illustrativehydrophilic spacer linkers include oxyalkylene groups, such as thefollowing formulae:

wherein n and r are integers and are each independently selected in eachinstance from 1 to about 5; and p is an integer selected from 1 to about4.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an independentlyselected integer from 1 to about 3; n is an integer from 1 to about 6, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one variation, the integer n is 3 or 4. In anothervariation, the integer p is 3 or 4. In another variation, the integer ris 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an independentlyselected integer from 1 to about 3; n is an integer from 2 to about 6, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one variation, the integer n is 3 or 4. In anothervariation, the integer p is 3 or 4. In another variation, the integer ris 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein m is an independently selected integer from 1 to about 3; n isan integer from 1 to about 6, p is an integer from 1 to about 5, and ris an integer selected from 1 to about 3. In one variation, the integern is 3 or 4. In another variation, the integer p is 3 or 4. In anothervariation, the integer r is 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein m is an independently selected integer from 1 to about 3; n isan integer from 2 to about 6, p is an integer from 1 to about 5, and ris an integer selected from 1 to about 3. In one variation, the integern is 3 or 4. In another variation, the integer p is 3 or 4. In anothervariation, the integer r is 1.

In another embodiment, the polyvalent linker L comprises one or more ofthe following fragments:

wherein m is an independently selected integer from 1 to about 3; n isan integer from 1 to about 6, p is an integer from 1 to about 5, and ris an integer selected from 1 to about 3. In one variation, the integern is 3 or 4. In another variation, the integer p is 3 or 4. In anothervariation, the integer r is 1.

In another embodiment, the polyvalent linker L comprises a combinationof backbone and branching side motifs such as is illustrated by thefollowing formulae

wherein n is an integer independently selected in each instance from 0to about 3. The above formula are intended to represent 4, 5, 6, andeven larger membered cyclic sugars. In addition, it is to be understoodthat the above formula may be modified to represent deoxy sugars, whereone or more of the hydroxy groups present on the formulae are replacedby hydrogen, alkyl, or amino. In addition, it is to be understood thatthe corresponding carbonyl compounds are contemplated by the aboveformulae, where one or more of the hydroxyl groups is oxidized to thecorresponding carbonyl. In addition, in this illustrative embodiment,the pyranose includes both carboxyl and amino functional groups and (a)can be inserted into the backbone and (b) can provide synthetic handlesfor branching side chains in variations of this embodiment. Any of thependant hydroxyl groups may be used to attach other chemical fragments,including additional sugars to prepare the correspondingoligosaccharides. Other variations of this embodiment are alsocontemplated, including inserting the pyranose or other sugar into thebackbone at a single carbon, i.e. a spiro arrangement, at a geminal pairof carbons, and like arrangements. For example, one or two ends of thelinker, or the drug D, or the binding ligand B may be connected to thesugar to be inserted into the backbone in a 1,1; 1,2; 1,3; 1,4; 2,3, orother arrangement.

In another embodiment, the hydrophilic spacer linkers described hereininclude are formed primarily from carbon, hydrogen, and nitrogen, andhave a carbon/nitrogen ratio of about 3:1 or less, or of about 2:1 orless. In one aspect, the hydrophilic linkers described herein include aplurality of amino functional groups.

In another embodiment, the polyvalent linker L comprises one or moreamino groups of the following formulae:

where n is an integer independently selected in each instance from 1 toabout 3. In one aspect, the integer n is independently 1 or 2 in eachinstance. In another aspect, the integer n is 1 in each instance.

In another embodiment, the polyvalent linker L comprises one or moresulfuric acid esters, such as an alkyl ester of sulfuric acid.Illustratively, the linker includes the following formula(e):

where n is an integer independently selected in each instance from 1 toabout 3. Illustratively, n is independently 1 or 2 in each instance.

It is understood, that in such polyhydroxyl, polyamino, carboxylic acid,sulfuric acid, and like linkers that include free hydrogens bound toheteroatoms, one or more of those free hydrogen atoms may be protectedwith the appropriate hydroxyl, amino, or acid protecting group,respectively, or alternatively may be blocked as the correspondingpro-drugs, the latter of which are selected for the particular use, suchas pro-drugs that release the parent drug under general or specificphysiological conditions.

In another embodiment, the polyvalent linker comprises one or more ofthe following divalent radicals:

wherein n is an integer from 2 to about 5, p is an integer from 1 toabout 5, and r is an integer from 1 to about 4, as described above.

It is to be further understood that in the foregoing embodiments, openpositions, such as (*) atoms are locations for attachment of the bindingligand (B) or any drug (D) to be delivered. In addition, it is to beunderstood that such attachment of either or both of B and any D may bedirect or through an intervening linker comprising one or more of theradicals described herein. In addition, (*) atoms may form releasablelinkers with any drug D, or other portion of the linker L.

In another embodiment, the hydrophilic spacer linker comprises one ormore carbohydrate containing or polyhydroxyl group containing linkers.In another embodiment, the hydrophilic spacer linker comprises at leastthree carbohydrate containing or polyhydroxyl group containing linkers.In another embodiment, the hydrophilic spacer linker comprises one ormore carbohydrate containing or polyhydroxyl group containing linkers,and one or more aspartic acids. In another embodiment, the hydrophilicspacer linker comprises one or more carbohydrate containing orpolyhydroxyl group containing linkers, and one or more glutamic acids.In another embodiment, the hydrophilic spacer linker comprises one ormore carbohydrate containing or polyhydroxyl group containing linkers,one or more glutamic acids, one or more aspartic acids, and one or morebeta amino alanines. In a series of variations, in each of the foregoingembodiments, the hydrophilic spacer linker also includes one or morecysteines. In another series of variations, in each of the foregoingembodiments, the hydrophilic spacer linker also includes at least onearginine.

In another embodiment, the polyvalent linker L includes a hydrophilicspacer linker comprising one or more divalent 1,4-piperazines that areincluded in the chain of atoms connecting at least one of the bindingligands (L) with at least one of the drugs (D). In one variation, thehydrophilic spacer linker includes one or more carbohydrate containingor polyhydroxyl group containing linkers. In another variation, thehydrophilic spacer linker includes one or more carbohydrate containingor polyhydroxyl group containing linkers and one or more aspartic acids.In another variation, the hydrophilic spacer linker includes one or morecarbohydrate containing or polyhydroxyl group containing linkers and oneor more glutamic acids. In a series of variations, in each of theforegoing embodiments, the hydrophilic spacer linker also includes oneor more cysteines. In another series of variations, in each of theforegoing embodiments, the hydrophilic spacer linker also includes atleast one arginine.

In another embodiment, the hydrophilic spacer linker comprises one ormore oligoamide hydrophilic spacers, such as but not limited toaminoethylpiperazinylacetamide.

In another embodiment, the polyvalent linker L includes a hydrophilicspacer linker comprising one or more triazole linked carbohydratecontaining or polyhydroxyl group containing linkers. In anotherembodiment, the hydrophilic spacer linker comprises one or more amidelinked carbohydrate containing or polyhydroxyl group containing linkers.In another embodiment, the hydrophilic spacer linker comprises one ormore PEG groups and one or more cysteines. In another embodiment, thehydrophilic spacer linker comprises one or more EDTE derivatives.

In another embodiment, the polyvalent linker L includes a divalentradical of the formula

wherein * indicates the point of attachment to a folate and ** indicatesthe point of attachment to a drug; and F and G are each independently 1,2, 3 or 4 are described.

In another embodiment, the polyvalent linker L includes a trivalentradical of the formula

wherein *, ** *** each indicate points of attachment to the folatereceptor binding moiety B, and the one or more drugs D. It is to beunderstood that when there are fewer drugs, *, **, *** are substitutedwith hydrogen or a heteroatom. F and G are each independently 1, 2, 3 or4; and W¹ is NH or O is described. In another aspect, m¹ is 0 or 1.

In any of the embodiments described herein heteroatom linkers can alsobe included in the polyvalent linker L, such as —NR¹R²—, oxygen, sulfur,and the formulae —(NHR¹NHR²)—, —SO—, —(SO₂)—, and —N(R³)O—, wherein R¹,R², and R³ are each independently selected from hydrogen, alkyl, aryl,arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl,substituted heteroaryl, and alkoxyalkyl. It is to be understood that theheteroatom linkers may be used to covalently attach any of the radicalsdescribed herein, including drug radicals D to the polyvalent linker,ligand radicals B to the polyvalent linker, or various di and polyvalentradicals that from the polyvalent linker L

Illustrative additional bivalent radicals that can be used to form partsof the linker are as follows.

In another embodiment, the polyvalent linker L is a releasable linker.

As used herein, the term “releasable linker” refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions when the compounds described herein are delivered to orinside of the target cell. The linker itself may include one or morecleavable, scissile, or breakable bond, or form one or more cleavable,scissile, or breakable bonds with the PSMA binding ligand (B), and/orwith one or more of the drugs (D). However, it is appreciated thatreleasable linkers described herein are advantageously not cleavable,scissile, or breakable until the conjugate containing the releasablelinker is at or near the intended target site. Accordingly, releasablelinkers described herein do not generally include those linkers thathave bonds that are substantially cleavable, scissile, or breakableunder non-target conditions, or in non-target tissues. Similarly,releasable linkers described herein do not include those linkers thatinclude bonds that are substantially only cleavable, scissile, orbreakable under non-physiological conditions.

The term releasable linker does not generally refer simply to a bondthat is labile in vivo, such as in serum, plasma, the gastrointestinaltract, or liver, unless those systems are the target for the cellsurface receptor binding ligand. However, after delivery and/orselective targeting, releasable linkers may be cleaved by any processthat includes at least one bond being broken in the linker or at thecovalent attachment of the linker to B or any D under physiologicalconditions, such as by having one or more pH-labile, acid-labile,base-labile, oxidatively labile, metabolically labile, biochemicallylabile, and/or enzyme-labile bonds. It is appreciated that suchphysiological conditions resulting in bond breaking do not necessarilyinclude a biological or metabolic process, and instead may include astandard chemical reaction, such as a hydrolysis reaction, for example,at physiological pH, or as a result of compartmentalization into acellular organelle such as an endosome having a lower pH than cytosolicpH.

It is understood that a cleavable bond can connect two adjacent atomswithin the releasable linker, and/or connect other linkers with B,and/or any D, as described herein, at any ends of the releasable linker.In the case where a cleavable bond connects two adjacent atoms withinthe releasable linker, following breakage of the bond, the releasablelinker is broken into two or more fragments. Alternatively, in the casewhere a cleavable bond is between the releasable linker and anothermoiety, such as an additional heteroatom, a spacer linker, anotherreleasable portion of the linker, any D, or B, following breakage of thebond, the releasable linker is separated from the other moiety. It is tobe understood that a linker is a releasable linker when if forms acleavable, scissile, or breakable bond with the one or more of the drugs(D) is capable of delivery of the one or more drugs (D) in a tracelessmanner, where the one or more drugs (D) do not include any residual partof the conjugate.

Illustrative radicals that themselves include a cleavable bond, or forma cleavable bond with B and/or any D hemiacetals and sulfur variationsthereof, acetals and sulfur variations thereof, hemiaminals, aminals,and the like, or which can be formed from methylene fragmentssubstituted with at least one heteroatom, such as 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, and the like. Illustrative releasablelinkers described herein include polyvalent linkers that includecarbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, haloalkylenecarbonyl, and the like.Illustrative releasable linkers described herein include polyvalentlinkers that include alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, and the like. Illustrative releasable linkersdescribed herein include oxycarbonyloxy, oxycarbonyloxyalkyl,sulfonyloxy, oxysulfonylalkyl, and the like. Illustrative releasablelinkers described herein include polyvalent linkers that includeiminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,carbonylcycloalkylideniminyl, and the like. Illustrative releasablelinkers described herein include polyvalent linkers that includealkylenethio, alkylenearylthio, and carbonylalkylthio, and the like.Each of the foregoing fragments is optionally substituted with asubstituent X², as defined herein.

The substituents X² can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the heteroatom linker canbe nitrogen, and the substituent X² and the heteroatom linker can betaken together with the releasable linker to which they are bound toform an heterocycle.

The heterocycles can be pyrrolidines, piperidines, oxazolidines,isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones,piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones,isothiazolidinones, and succinimides.

Illustrative releasable linkers include ketals, acetals, hemiaminals,and aminals formed from methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl radicals, esters and amides formed fromcarbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and haloalkylenecarbonyl radicals,oxysilanes and aminosilanes formed from alkylene(dialkylsilyl),alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl,(alkylarylsilyl)aryl, and (diarylsilyl)aryl radicals, oxycarbonyloxy,oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl,carbonylalkylideniminyl, iminocycloalkylidenyl,carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, andcarbonylalkylthio radicals, each of which is optionally substituted.

Further illustrative releasable linkers include hydrazones,acylhydrazones orthoformates, and carbamoyl derivatives.

Further illustrative releasable linkers include disulfides and activatedthioethers.

In any of the embodiments described herein, the releasable linker mayinclude oxygen bonded to methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl to form an acetal or ketal, wherein eachof the fragments is optionally substituted with a substituent X², asdefined herein. Alternatively, the methylene or alkylene is substitutedwith an optionally-substituted aryl.

In any of the embodiments described herein, the releasable linker mayinclude nitrogen bonded to methylene, I-alkoxyalkylene,1-alkoxycycloalkylene, I-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl to form a hemiamninal ether or aminal,wherein each of the fragments is optionally substituted with asubstituent X², as defined herein. Alternatively, the methylene oralkylene is substituted with an optionally-substituted aryl.

In any of the embodiments described herein, the releasable linker mayinclude oxygen bonded to sulfonylalkyl to form an alkylsulfonate.

In any of the embodiments described herein, the releasable linker mayinclude nitrogen bonded to iminoalkylidenyl, carbonylalkylideniminyl,iminocycloalkylidenyl, and carbonylcycloalkylideniminyl to form anhydrazone, each of which is optionally substituted with a substituentX², as defined herein. In an alternate configuration, the hydrazone maybe acylated with a carboxylic acid derivative, an orthoformatederivative, or a carbamoyl derivative to form releasable linkerscontaining various acylhydrazones.

In any of the embodiments described herein, the releasable linker mayinclude oxygen bonded to alkylene(dialkylsilyl),alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl,(alkylarylsilyl)aryl, and (diarylsilyl)aryl to form a silanol, each ofwhich is optionally substituted with a substituent X², as definedherein.

In any of the embodiments described herein, the releasable linker mayinclude nitrogen bonded to carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to forman amide, or alternatively an amide with a drug nitrogen.

In any of the embodiments described herein, the releasable linker mayinclude oxygen bonded to carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to forman ester, or alternatively an ester with drug oxygen.

It is to be understood that the bivalent spacer linkers may be combinedin any chemically relevant way, either directly or via an interveningheteroatom to construct the releasable linkers described herein. It isfurther understood that the nature of the arrangement of spacer andheteroatom linkers defines where the releasable linker will cleave invivo. For example, two spacer linkers that terminate in a sulfur atomwhen combined form a disulfide, which is the cleavable bond in thereleasable linker formed thereby.

For example, in another embodiment, the polyvalent linker comprises a3-thiosuccinimid-1-ylalkyloxymethyloxy moiety, where the methyl isoptionally substituted with alkyl or substituted aryl.

In another embodiment, the polyvalent linker comprises a3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms anacylaziridine with the drug.

In another embodiment, the polyvalent linker comprises a1-alkoxycycloalkylenoxy moiety.

In another embodiment, the polyvalent linker comprises analkyleneaminocarbonyl(dicarboxylarylene)carboxylate.

In another embodiment, the polyvalent linker comprises adithioalkylcarbonylhydrazide, where the hydrazide forms an hydrazonewith the drug.

In another embodiment, the polyvalent linker comprises a3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide forms ahydrazone with the drug.

In another embodiment, the polyvalent linker comprises a3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where thedisubstituted silyl is substituted with alkyl or optionally substitutedaryl.

In another embodiment, the polyvalent linker comprises a plurality ofspacer linkers selected from the group consisting of the naturallyoccurring amino acids and stereoisomers thereof.

In another embodiment, the polyvalent linker comprises a2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with thedrug.

In another embodiment, the polyvalent linker comprises a2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate withthe drug and the aryl is optionally substituted.

In another embodiment, the polyvalent linker comprises a4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate withthe drug, and the aryl is optionally substituted.

In another embodiment, the polyvalent linker comprises a3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylideneforms an hydrazone with the drug, each alkyl is independently selected,and the oxyalkyloxy is optionally substituted with alkyl or optionallysubstituted aryl.

In another embodiment, the polyvalent linker comprises a2-dithioalkyloxycarbonylhydrazide.

In another embodiment, the polyvalent linker comprises a 2- or3-dithioalkylamino, where the amino forms a vinylogous amide with thedrug.

In another embodiment, the polyvalent linker comprises a2-dithioalkylamino, where the amino forms a vinylogous amide with thedrug, and the alkyl is ethyl.

In another embodiment, the polyvalent linker comprises a 2- or3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug.

In another embodiment, the polyvalent linker comprises a2-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug. In another aspect, the alkyl is ethyl.

In another embodiment, the polyvalent linker comprises a2-dithioalkyloxycarbonyl, where the carbonyl forms a carbamate with thedrug. In another aspect, the alkyl is ethyl.

In another embodiment, the polyvalent linker comprises a2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or acarbamoylaziridine with the drug.

In another embodiment, the polyvalent linker comprises a4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or acarbamoylaziridine with the drug.

In another embodiment, the polyvalent linkers described herein comprisedivalent radicals of the formulae

where n is an integer selected from 1 to about 4; R^(a) and R^(b) areeach independently selected from the group consisting of hydrogen andalkyl, including lower alkyl such as C₁-C₄ alkyl that are optionallybranched; or R^(a) and R^(b) are taken together with the attached carbonatom to form a carbocyclic ring; R is an optionally substituted alkylgroup, an optionally substituted acyl group, or a suitably selectednitrogen protecting group; and (*) indicates points of attachment forthe drug, vitamin, imaging agent, diagnostic agent, other bivalentlinkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein comprisedivalent radicals of the formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherbivalent linkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein comprisedivalent radicals of the formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherdivalent linkers, or other parts of the conjugate.

In another embodiment, the compounds described herein comprise one ormore radicals linkers of selected from the formulae:

wherein X is NH, O, or S.

In another embodiment, the polyvalent linkers herein described comprisea radical having the formula:

Another embodiment, the polyvalent linkers described herein comprise aradical of having the formula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and the symbol (*) indicates points of attachment. It isappreciated that other substituents may be present on the aryl ring, thebenzyl carbon, the alkanoic acid, or the methylene bridge, including butnot limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and the like.

In another embodiment, the polyvalent linkers described herein compriseradicals selected from carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1 alkylenesuccinimid-3-yl,1 (carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of saidspacer linkers is optionally substituted with one or more substituentsX¹;

wherein each substituent X¹ is independently selected from the groupconsisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl,sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy,carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl,wherein R⁴ and R⁵ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide,and wherein R⁶ and R⁷ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide.

It is to be understood that the compounds described herein may containone or more chiral centers, or may otherwise be capable of existing asmultiple stereoisomers. It is to be understood that in one embodiment,the invention described herein is not limited to any particularstereochemical requirement, and that the compounds, and compositions,methods, uses, and medicaments that include them may be optically pure,or may be any of a variety of stereoisomeric mixtures, including racemicand other mixtures of enantiomers, other mixtures of diastereomers, andthe like. It is also to be understood that such mixtures ofstereoisomers may include a single stereochemical configuration at oneor more chiral centers, while including mixtures of stereochemicalconfiguration at one or more other chiral centers.

Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration at one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

In each of the foregoing and each of the following embodiments, it isalso to be understood that the formulae include and represent not onlyall pharmaceutically acceptable salts of the compounds, but also includeany and all hydrates and/or solvates of the compound formulae. It isappreciated that certain functional groups, such as the hydroxy, amino,and like groups form complexes and/or coordination compounds with waterand/or various solvents, in the various physical forms of the compounds.Accordingly, the above formulae are to be understood to be a descriptionof such hydrates and/or solvates, including pharmaceutically acceptablesolvates.

In each of the foregoing and each of the following embodiments, it isalso to be understood that the formulae include and represent eachpossible isomer, such as stereoisomers and geometric isomers, bothindividually and in any and all possible mixtures. In each of theforegoing and each of the following embodiments, it is also to beunderstood that the formulae include and represent any and allcrystalline forms, partially crystalline forms, and non crystallineand/or amorphous forms, and co-crystals of the compounds.

In another embodiment, the compounds described herein can beinternalized into the targeted pathogenic cells by binding to PSMA. Inparticular, PSMA selectively and/or specifically binds the conjugate,and internalization can occur, for example, through PSMA-mediatedendocytosis. Once internalized, conjugates containing a releasablelinker can complete delivery of the drug to the interior of the targetcell. Without being bound by theory, it is believed herein that in thosecases where the drug is toxic to normal cells or tissues, such adelivery system can decrease toxicity against those non-target cells andtissues because the releasable linker remains substantially orcompletely intact until the compounds described herein are delivered tothe target cells. Accordingly, the compounds described herein actintracellularly by delivering the drug to an intracellular biochemicalprocess, which in turn decreases the amount of unconjugated drugexposure to the host animal's healthy cells and tissues.

The conjugates described herein can be used for both human clinicalmedicine and veterinary applications. Thus, the host animal harboringthe population of pathogenic cells and treated with the compoundsdescribed herein can be human or, in the case of veterinaryapplications, can be a laboratory, agricultural, domestic, or wildanimal. The present invention can be applied to host animals including,but not limited to, humans, laboratory animals such rodents (e.g., mice,rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animalssuch as dogs, cats, and rabbits, agricultural animals such as cows,horses, pigs, sheep, goats, and wild animals in captivity such as bears,pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas,dolphins, and whales.

The drug delivery conjugate compounds described herein can beadministered in a combination therapy with any other known drug whetheror not the additional drug is targeted. Illustrative additional drugsinclude, but are not limited to, peptides, oligopeptides, retro-inversooligopeptides, proteins, protein analogs in which at least onenon-peptide linkage replaces a peptide linkage, apoproteins,glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids andtheir derivatives, receptors and other membrane proteins, antigens andantibodies thereto, haptens and antibodies thereto, hormones, lipids,phospholipids, liposomes, toxins, antibiotics, analgesics,bronchodilators, beta-blockers, antimicrobial agents, antihypertensiveagents, cardiovascular agents including antiarrhythmics, cardiacglycosides, antianginals, vasodilators, central nervous system agentsincluding stimulants, psychotropics, antimanics, and depressants,antiviral agents, antihistamines, cancer drugs includingchemotherapeutic agents, tranquilizers, anti-depressants, H-2antagonists, anticonvulsants, antinauseants, prostaglandins andprostaglandin analogs, muscle relaxants, anti-inflammatory substances,stimulants, decongestants, antiemetics, diuretics, antispasmodics,antiasthmatics, anti-Parkinson agents, expectorants, cough suppressants,mucolytics, and mineral and nutritional additives.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched. As used herein, the term “alkenyl” and “alkynyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond or triple bond, respectively. It is tobe understood that alkynyl may also include one or more double bonds. Itis to be further understood that in certain embodiments, alkyl isadvantageously of limited length, including C₁-C₂₄, C₁-C₁₂, C₁-C₈,C₁-C₆, and C₁-C₄, and C₂-C₂₄, C₂-C₁₂, C₂-C₈. C₂-C₆, and C₂-C₄, and thelike Illustratively, such particularly limited length alkyl groups,including C₁-C₈, C₁-C₆, and C₁-C₄, and C₂-C₈, C₂-C₆, and C₂-C₄, and thelike may be referred to as lower alkyl. It is to be further understoodthat in certain embodiments alkenyl and/or alkynyl may each beadvantageously of limited length, including C₂-C₂₄, C₂-C₁₂, C₂-C₈,C₂-C₆, and C₂—C₄, and C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₃-C₄, and thelike. Illustratively, such particularly limited length alkenyl and/oralkynyl groups, including C₂-C₈, C₂-C₆, and C₂-C₄, and C₃-C₈, C₃-C₆, andC₃-C₄, and the like may be referred to as lower alkenyl and/or alkynyl.It is appreciated herein that shorter alkyl, alkenyl, and/or alkynylgroups may add less lipophilicity to the compound and accordingly willhave different pharmacokinetic behavior. In embodiments of the inventiondescribed herein, it is to be understood, in each case, that therecitation of alkyl refers to alkyl as defined herein, and optionallylower alkyl. In embodiments of the invention described herein, it is tobe understood, in each case, that the recitation of alkenyl refers toalkenyl as defined herein, and optionally lower alkenyl. In embodimentsof the invention described herein, it is to be understood, in each case,that the recitation of alkynyl refers to alkynyl as defined herein, andoptionally lower alkynyl. Illustrative alkyl, alkenyl, and alkynylgroups are, but not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl,neopentyl, hexyl, heptyl, octyl, and the like, and the correspondinggroups containing one or more double and/or triple bonds, or acombination thereof.

As used herein, the term “alkylene” includes a divalent chain of carbonatoms, which is optionally branched. As used herein, the term“alkenylene” and “alkynylene” includes a divalent chain of carbon atoms,which is optionally branched, and includes at least one double bond ortriple bond, respectively. It is to be understood that alkynylene mayalso include one or more double bonds. It is to be further understoodthat in certain embodiments, alkylene is advantageously of limitedlength, including C₁-C₂₄, C₁-C₁₂, C₁-C₈, C₁-C₆, and C₁-C₄, and C₂-C₂₄,C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄, and the like. Illustratively, suchparticularly limited length alkylene groups, including C₁-C₈, C₁-C₆, andC₁-C₄, and C₂-C₈, C₂-C₆, and C₂-C₄, and the like may be referred to aslower alkylene. It is to be further understood that in certainembodiments alkenylene and/or alkynylene may each be advantageously oflimited length, including C₂-C₂₄, C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄, andC₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₃-C₄, and the like. Illustratively,such particularly limited length alkenylene and/or alkynylene groups,including C₂-C₈, C₂-C₆, and C₂-C₄, and C₃-C₈, C₃-C₆, and C₃-C₄, and thelike may be referred to as lower alkenylene and/or alkynylene. It isappreciated herein that shorter alkylene, alkenylene, and/or alkynylenegroups may add less lipophilicity to the compound and accordingly willhave different pharmacokinetic behavior. In embodiments of the inventiondescribed herein, it is to be understood, in each case, that therecitation of alkylene, alkenylene, and alkynylene refers to alkylene,alkenylene, and alkynylene as defined herein, and optionally loweralkylene, alkenylene, and alkynylene. Illustrative alkyl groups are, butnot limited to, methylene, ethylene, n-propylene, isopropylene,n-butylene, isobutylene, sec-butylene, pentylene, 1,2-pentylene,1,3-pentylene, hexylene, heptylene, octylene, and the like.

As used herein, the term “cycloalkyl” includes a chain of carbon atoms,which is optionally branched, where at least a portion of the chain incyclic. It is to be understood that cycloalkylalkyl is a subset ofcycloalkyl. It is to be understood that cycloalkyl may be polycyclic.Illustrative cycloalkyl include, but are not limited to, cyclopropyl,cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl,adamantyl, and the like. As used herein, the term “cycloalkenyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond, where at least a portion of the chainin cyclic. It is to be understood that the one or more double bonds maybe in the cyclic portion of cycloalkenyl and/or the non-cyclic portionof cycloalkenyl. It is to be understood that cycloalkenylalkyl andcycloalkylalkenyl are each subsets of cycloalkenyl. It is to beunderstood that cycloalkyl may be polycyclic. Illustrative cycloalkenylinclude, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl,cycloheptenylpropenyl, and the like. It is to be further understood thatchain forming cycloalkyl and/or cycloalkenyl is advantageously oflimited length, including C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₅-C₆. It isappreciated herein that shorter alkyl and/or alkenyl chains formingcycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicityto the compound and accordingly will have different pharmacokineticbehavior.

As used herein, the term “heteroalkyl” includes a chain of atoms thatincludes both carbon and at least one heteroatom, and is optionallybranched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur.In certain variations, illustrative heteroatoms also include phosphorus,and selenium. As used herein, the term “cycloheteroalkyl” includingheterocyclyl and heterocycle, includes a chain of atoms that includesboth carbon and at least one heteroatom, such as heteroalkyl, and isoptionally branched, where at least a portion of the chain is cyclic.Illustrative heteroatoms include nitrogen, oxygen, and sulfur. Incertain variations, illustrative heteroatoms also include phosphorus,and selenium. Illustrative cycloheteroalkyl include, but are not limitedto, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term “aryl” includes monocyclic and polycyclicaromatic carbocyclic groups, each of which may be optionallysubstituted. Illustrative aromatic carbocyclic groups described hereininclude, but are not limited to, phenyl, naphthyl, and the like. As usedherein, the term “heteroaryl” includes aromatic heterocyclic groups,each of which may be optionally substituted. Illustrative aromaticheterocyclic groups include, but are not limited to, pyridinyl,pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl,quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl,benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl,benzisothiazolyl, and the like.

As used herein, the term “amino” includes the group NH₂, alkylamino, anddialkylamino, where the two alkyl groups in dialkylamino may be the sameor different, i.e. alkylalkylamino. Illustratively, amino includesmethylamino, ethylamino, dimethylamino, methylethylamino, and the like.In addition, it is to be understood that when amino modifies or ismodified by another term, such as aminoalkyl, or acylamino, the abovevariations of the term amino are included therein. Illustratively,aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl,dimethylaminoalkyl, methylethylaminoalkyl, and the like. Illustratively,acylamino includes acylmethylamino, acylethylamino, and the like.

As used herein, the term “amino and derivatives thereof” includes aminoas described herein, and alkylamino, alkenylamino, alkynylamino,heteroalkylamino, heteroalkenylamino, heteroalkynylamino,cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino,cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino,arylalkynylamino, heteroarylamino, heteroarylalkylamino,heteroarylalkenylamino, heteroarylalkynylamino, acylamino, and the like,each of which is optionally substituted. The term “amino derivative”also includes urea, carbamate, and the like.

As used herein, the term “amino acid” refers generally to beta, gamma,and longer amino acids, such as amino acids of the formula:

—N(R)—(CR′R″)_(q)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornitine, threonine, and thelike.

As used herein, the term “amino acid derivative” generally refers to anamino acid as defined herein where either, or both, the amino groupand/or the side chain is substituted. Illustrative amino acidderivatives include prodrugs and protecting groups of the amino groupand/or the side chain, such as amine, amide, hydroxy, carboxylic acid,and thio prodrugs and protecting groups. Additional Illustrative aminoacid derivatives include substituted variations of the amino acid asdescribed herein, such as, but not limited to, ethers and esters ofhydroxy groups, amides, carbamates, and ureas of amino groups, esters,amides, and cyano derivatives of carboxylic acid groups, and the like.

As used herein, the term “hydroxy and derivatives thereof” includes OH,and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy,heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy,cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy,arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkenyloxy,heteroarylalkynyloxy, acyloxy, and the like, each of which is optionallysubstituted. The term “hydroxy derivative” also includes carbamate, andthe like.

As used herein, the term “thio and derivatives thereof” includes SH, andalkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio,heteroalkynylthio, cycloalkylthio, cycloalkenylthio,cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio,arylalkenylthio, arylalkynylthio, heteroarylthio, heteroarylalkylthio,heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the like,each of which is optionally substituted. The term “thio derivative” alsoincludes thiocarbamate, and the like.

As used herein, the term “acyl” includes formyl, and alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl,heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of which isoptionally substituted.

As used herein, the term “carbonyl and derivatives thereof” includes thegroup C(O), C(S), C(NH) and substituted amino derivatives thereof.

As used herein, the term “carboxylic acid and derivatives thereof”includes the group CO₂H and salts thereof, and esters and amidesthereof, and CN.

As used herein, the term “sulfinic acid or a derivative thereof”includes SO₂H and salts thereof, and esters and amides thereof.

As used herein, the term “sulfonic acid or a derivative thereof”includes SO₃H and salts thereof, and esters and amides thereof.

As used herein, the term “sulfonyl” includes alkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, heteroalkylsulfonyl,heteroalkenylsulfonyl, heteroalkynylsulfonyl, cycloalkylsulfonyl,cycloalkenylsulfonyl, cycloheteroalkylsulfonyl,cycloheteroalkenylsulfonyl, arylsulfonyl, arylalkylsulfonyl,arylalkenylsulfonyl, arylalkynylsulfonyl, heteroarylsulfonyl,heteroarylalkylsulfonyl, heteroarylalkenylsulfonyl,heteroarylalkynylsulfonyl, acylsulfonyl, and the like, each of which isoptionally substituted.

As used herein, the term “phosphinic acid or a derivative thereof”includes P(R)O₂H and salts thereof, and esters and amides thereof, whereR is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl,heteroalkenyl, cycloheteroalkyl, cycloheteroalkenyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl, each of which is optionally substituted.

As used herein, the term “phosphonic acid or a derivative thereof”includes PO₃H₂ and salts thereof, and esters and amides thereof.

As used herein, the term “hydroxylamino and derivatives thereof”includes NHOH, and alkyloxylNH alkenyloxylNH alkynyloxylNHheteroalkyloxylNH heteroalkenyloxylNH heteroalkynyloxylNHcycloalkyloxylNH cycloalkenyloxylNH cycloheteroalkyloxylNHcycloheteroalkenyloxylNH aryloxylNH arylalkyloxylNH arylalkenyloxylNHarylalkynyloxylNH heteroaryloxylNH heteroarylalkyloxylNHheteroarylalkenyloxylNH heteroarylalkynyloxylNH acyloxy, and the like,each of which is optionally substituted.

As used herein, the term “hydrazino and derivatives thereof” includesalkylNHNH, alkenylNHNH, alkynylNHNH, heteroalkylNHNH, heteroalkenylNHNH,heteroalkynylNHNH, cycloalkylNHNH, cycloalkenylNHNH,cycloheteroalkylNHNH, cycloheteroalkenylNHNH, arylNHNH, arylalkylNHNH,arylalkenylNHNH, arylalkynylNHNH, heteroarylNHNH, heteroarylalkylNHNH,heteroarylalkenylNHNH, heteroarylalkynylNHNH, acylNHNH, and the like,each of which is optionally substituted.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonicacids and derivatives thereof, carboxylic acids and derivatives thereof,and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl,haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is alsooptionally substituted.

As used herein, the terms “optionally substituted aryl” and “optionallysubstituted heteroaryl” include the replacement of hydrogen atoms withother functional groups on the aryl or heteroaryl that is optionallysubstituted. Such other functional groups illustratively include, butare not limited to, amino, hydroxy, halo, thio, alkyl, haloalkyl,heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids andderivatives thereof, carboxylic acids and derivatives thereof, and thelike. Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid isoptionally substituted.

Illustrative substituents include, but are not limited to, a radical—(CH₂)_(x)Z^(X), where x is an integer from 0-6 and Z^(X) is selectedfrom halogen, hydroxy, alkanoyloxy, including C₁-C₆ alkanoyloxy,optionally substituted aroyloxy, alkyl, including C₁-C₆ alkyl, alkoxy,including C₁-C₆ alkoxy, cycloalkyl, including C₃-C₈ cycloalkyl,cycloalkoxy, including C₃-C₈ cycloalkoxy, alkenyl, including C₂-C₆alkenyl, alkynyl, including C₂-C₆ alkynyl, haloalkyl, including C₁-C₆haloalkyl, haloalkoxy, including C₁-C₆ haloalkoxy, halocycloalkyl,including C₃-C₈ halocycloalkyl, halocycloalkoxy, including C₃-C₈halocycloalkoxy, amino. C₁-C₆ alkylamino, (C₁-C₆ alkyl)(C₁-C₆alkyl)amino, alkylcarbonylamino, N—(C₁-C₆ alkyl)alkylcarbonylamino,aminoalkyl, C₁-C₆ alkylaminoalkyl, (C₁-C₆ alkyl)(C₁-C₆ alkyl)aminoalkyl,alkylcarbonylaminoalkyl, N—(C₁-C₆ alkyl)alkylcarbonylaminoalkyl, cyano,and nitro; or Z^(X) is selected from —CO₂R⁴ and —CONR⁵R⁶, where R⁴, R⁵,and R⁶ are each independently selected in each occurrence from hydrogen,C₁-C₆ alkyl, aryl-C₁-C₆ alkyl, and heteroaryl-C₁-C₆ alkyl.

As used herein, the term “leaving group” refers to a reactive functionalgroup that generates an electrophilic site on the atom to which it isattached such that nucleophiles may be added to the electrophilic siteon the atom. Illustrative leaving groups include, but are not limitedto, halogens, optionally substituted phenols, acyloxy groups, sulfonoxygroups, and the like. It is to be understood that such leaving groupsmay be on alkyl, acyl, and the like. Such leaving groups may also bereferred to herein as activating groups, such as when the leaving groupis present on acyl. In addition, conventional peptide, amide, and estercoupling agents, such as but not limited to PyBop. BOP-Cl, BOP,pentafluorophenol, isobutylchloroformate, and the like, form variousintermediates that include a leaving group, as defined herein, on acarbonyl group.

As used herein the term “radical” with reference to, for example, thePSMA binding or targeting ligand, and/or the independently selecteddrug, refers to a PSMA binding or targeting ligand, and/or anindependently selected drug, as described herein, where one or moreatoms or groups, such as a hydrogen atom, or an alkyl group on aheteroatom, and the like, is removed to provide a radical forconjugation to the polyvalent linker L.

The term “prodrug” as used herein generally refers to any compound thatwhen administered to a biological system generates a biologically activecompound as a result of one or more spontaneous chemical reaction(s),enzyme-catalyzed chemical reaction(s), and/or metabolic chemicalreaction(s), or a combination thereof. In vivo, the prodrug is typicallyacted upon by an enzyme (such as esterases, amidases, phosphatases, andthe like), simple biological chemistry, or other process in vivo toliberate or regenerate the more pharmacologically active drug. Thisactivation may occur through the action of an endogenous host enzyme ora non-endogenous enzyme that is administered to the host preceding,following, or during administration of the prodrug. Additional detailsof prodrug use are described in U.S. Pat. No. 5,627,165; and Pathalk etal., Enzymic protecting group techniques in organic synthesis,Stereosel. Biocatal. 775-797 (2000). It is appreciated that the prodrugis advantageously converted to the original drug as soon as the goal,such as targeted delivery, safety, stability, and the like is achieved,followed by the subsequent rapid elimination of the released remains ofthe group forming the prodrug.

Prodrugs may be prepared from the compounds described herein byattaching groups that ultimately cleave in vivo to one or morefunctional groups present on the compound, such as —OH—, —SH, —CO₂H,—NR₂. Illustrative prodrugs include but are not limited to carboxylateesters where the group is alkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as estersof hydroxyl, thiol and amines where the group attached is an acyl group,an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrativeesters, also referred to as active esters, include but are not limitedto 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such asacetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl, andthe like; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl,β-ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl groups,including di-lower alkylamino alkyl groups, such as dimethylaminomethyl,dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like;2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and lactonegroups such as phthalidyl, dimethoxyphthalidyl, and the like.

Further illustrative prodrugs contain a chemical moiety, such as anamide or phosphorus group functioning to increase solubility and/orstability of the compounds described herein. Further illustrativeprodrugs for amino groups include, but are not limited to.(C₃-C₂₀)alkanoyl; halo-(C₃-C₂₀)alkanoyl; (C₃-C₂₀)alkenoyl;(C₄-C₇)cycloalkanoyl; (C₃-C₆)-cycloalkyl(C₂-C₁₆)alkanoyl; optionallysubstituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1to 3 substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with one or more of 1 to 3halogen atoms; optionally substituted aryl(C₂-C₁₆)alkanoyl andoptionally substituted heteroaryl(C₂-C₁₆)alkanoyl, such as the aryl orheteroaryl radical being unsubstituted or substituted by 1 to 3substituents selected from the group consisting of halogen, (C₁-C₃)alkyland (C₁-C₃)alkoxy, each of which is optionally further substituted with1 to 3 halogen atoms; and optionally substituted heteroarylalkanoylhaving one to three heteroatoms selected from O, S and N in theheteroaryl moiety and 2 to 10 carbon atoms in the alkanoyl moiety, suchas the heteroaryl radical being unsubstituted or substituted by 1 to 3substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl, and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with 1 to 3 halogen atoms. Thegroups illustrated are exemplary, not exhaustive, and may be prepared byconventional processes.

It is understood that the prodrugs themselves may not possesssignificant biological activity, but instead undergo one or morespontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s),and/or metabolic chemical reaction(s), or a combination thereof afteradministration in vivo to produce the compound described herein that isbiologically active or is a precursor of the biologically activecompound. However, it is appreciated that in some cases, the prodrug isbiologically active. It is also appreciated that prodrugs may oftenserves to improve drug efficacy or safety through improved oralbioavailability, pharmacodynamic half-life, and the like. Prodrugs alsorefer to derivatives of the compounds described herein that includegroups that simply mask undesirable drug properties or improve drugdelivery. For example, one or more compounds described herein mayexhibit an undesirable property that is advantageously blocked orminimized may become pharmacological, pharmaceutical, or pharmacokineticbarriers in clinical drug application, such as low oral drug absorption,lack of site specificity, chemical instability, toxicity, and poorpatient acceptance (bad taste, odor, pain at injection site, and thelike), and others. It is appreciated herein that a prodrug, or otherstrategy using reversible derivatives, can be useful in the optimizationof the clinical application of a drug.

It is to be understood that in every instance disclosed herein, therecitation of a range of integers for any variable describes the recitedrange, every individual member in the range, and every possible subrangefor that variable. For example, the recitation that n is an integer from0 to 8, describes that range, the individual and selectable values of 0,1, 2, 3, 4, 5, 6, 7, and 8, such as n is 0, or n is 1, or n is 2, etc.In addition, the recitation that n is an integer from 0 to 8 alsodescribes each and every subrange, each of which may for the basis of afurther embodiment, such as n is an integer from 1 to 8, from 1 to 7,from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from variousamorphous, non-amorphous, partially crystalline, crystalline, and/orother morphological forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from varioushydrates and/or solvates of the compounds described herein. Accordingly,such pharmaceutical compositions that recite compounds described hereinare to be understood to include each of, or any combination of, thevarious morphological forms and/or solvate or hydrate forms of thecompounds described herein. In addition, it is to be understood that thecompositions may be prepared from various co-crystals of the compoundsdescribed herein.

Illustratively, compositions may include one or more carriers, diluents,and/or excipients. The compounds described herein, or compositionscontaining them, may be formulated in a therapeutically effective amountin any conventional dosage forms appropriate for the methods describedherein. The compounds described herein, or compositions containing them,including such formulations, may be administered by a wide variety ofconventional routes for the methods described herein, and in a widevariety of dosage formats, utilizing known procedures (see generally,Remington: The Science and Practice of Pharmacy, (21^(st) ed., 2005)).

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill.

It is also appreciated that the therapeutically effective amount,whether referring to monotherapy or combination therapy, isadvantageously selected with reference to any toxicity, or otherundesirable side effect, that might occur during administration of oneor more of the compounds described herein. Further, it is appreciatedthat the co-therapies described herein may allow for the administrationof lower doses of compounds that show such toxicity, or otherundesirable side effect, where those lower doses are below thresholds oftoxicity or lower in the therapeutic window than would otherwise beadministered in the absence of a cotherapy.

In addition to the illustrative dosages and dosing protocols describedherein, it is to be understood that an effective amount of any one or amixture of the compounds described herein can be readily determined bythe attending diagnostician or physician by the use of known techniquesand/or by observing results obtained under analogous circumstances. Indetermining the effective amount or dose, a number of factors areconsidered by the attending diagnostician or physician, including, butnot limited to the species of mammal, including human, its size, age,and general health, the specific disease or disorder involved, thedegree of or involvement or the severity of the disease or disorder, theresponse of the individual patient, the particular compoundadministered, the mode of administration, the bioavailabilitycharacteristics of the preparation administered, the dose regimenselected, the use of concomitant medication, and other relevantcircumstances.

The dosage of each compound of the claimed combinations depends onseveral factors, including: the administration method, the condition tobe treated, the severity of the condition, whether the condition is tobe treated or prevented, and the age, weight, and health of the personto be treated. Additionally, pharmacogenomic (the effect of genotype onthe pharmacokinetic, pharmacodynamic or efficacy profile of atherapeutic) information about a particular patient may affect thedosage used.

It is to be understood that in the methods described herein, theindividual components of a co-administration, or combination can beadministered by any suitable means, contemporaneously, simultaneously,sequentially, separately or in a single pharmaceutical formulation.Where the co-administered compounds or compositions are administered inseparate dosage forms, the number of dosages administered per day foreach compound may be the same or different. The compounds orcompositions may be administered via the same or different routes ofadministration. The compounds or compositions may be administeredaccording to simultaneous or alternating regimens, at the same ordifferent times during the course of the therapy, concurrently individed or single forms.

The term “administering” as used herein includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxicpharmaceutically-acceptable carriers, adjuvants, and/or vehicles.

Illustrative formats for oral administration include tablets, capsules,elixirs, syrups, and the like.

Illustrative routes for parenteral administration include intravenous,intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal,intramuscular and subcutaneous, as well as any other art recognizedroute of parenteral administration.

Illustratively, administering includes local use, such as whenadministered locally to the site of disease, injury, or defect, or to aparticular organ or tissue system. Illustrative local administration maybe performed during open surgery, or other procedures when the site ofdisease, injury, or defect is accessible. Alternatively, localadministration may be performed using parenteral delivery where thecompound or compositions described herein are deposited locally to thesite without general distribution to multiple other non-target sites inthe patient being treated. It is further appreciated that localadministration may be directly in the injury site, or locally in thesurrounding tissue. Similar variations regarding local delivery toparticular tissue types, such as organs, and the like, are alsodescribed herein. Illustratively, compounds may be administered directlyto the nervous system including, but not limited to, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracistemal,intraspinal and/or peri-spinal routes of administration by delivery viaintracranial or intravertebral needles and/or catheters with or withoutpump devices.

Depending upon the disease as described herein, the route ofadministration and/or whether the compounds and/or compositions areadministered locally or systemically, a wide range of permissibledosages are contemplated herein, including doses falling in the rangefrom about 1 μg/kg to about 1 g/kg. The dosages may be single ordivided, and may administered according to a wide variety of protocols,including q.d., b.i.d., t.i.d., or even every other day, once a week,once a month, once a quarter, and the like. In each of these cases it isunderstood that the therapeutically effective amounts described hereincorrespond to the instance of administration, or alternatively to thetotal daily, weekly, month, or quarterly dose, as determined by thedosing protocol.

In making the pharmaceutical compositions of the compounds describedherein, a therapeutically effective amount of one or more compounds inany of the various forms described herein may be mixed with one or moreexcipients, diluted by one or more excipients, or enclosed within such acarrier which can be in the form of a capsule, sachet, paper, or othercontainer. Excipients may serve as a diluent, and can be solid,semi-solid, or liquid materials, which act as a vehicle, carrier ormedium for the active ingredient. Thus, the formulation compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders. The compositions may contain anywhere from about 0.1% to about99.9% active ingredients, depending upon the selected dose and dosageform.

The effective use of the compounds, compositions, and methods describedherein for treating or ameliorating diseases caused by pathogenic cellsexpressing PSMA may be based upon animal models, such as murine, canine,porcine, and non-human primate animal models of disease. For example, itis understood that prostate cancer in humans may be characterized by aloss of function, and/or the development of symptoms, each of which maybe elicited in animals, such as mice, and other surrogate test animals.In particular the mouse models described herein where cancer cells, suchas LNCaP cells are subcutaneously implanted may be used to evaluate thecompounds, the methods of treatment, and the pharmaceutical compositionsdescribed herein to determine the therapeutically effective amountsdescribed herein.

The compounds, linkers, intermediates, and conjugates described hereinmay be prepared using conventional processes, including those describedin International Patent Publication Nos. WO 2009/002993, WO 2004/069159,WO 2007/022494, and WO 2006/012527, and U.S. patent application Ser. No.13/837,539 (filed Mar. 15, 2013). The disclosures of each of theforegoing are herein incorporated by reference in their entirety.

Each of the publications cited herein is incorporated herein byreference.

The following examples further illustrate specific embodiments of theinvention; however, the following illustrative examples should not beinterpreted in any way to limit the invention.

EXAMPLES

Example. Compound 104

In a 250 mL round-bottom flask, H-Glu(OtBu)-OtBu.HCl (1) (4.83 g, 16.3mmol) and 4-nitrophenyl chloroformate (102) (3.47 g, 17.2 mmol) weredissolved in dichloromethane (50 mL) and stirred in an ice bath underargon. Diisopropylethylamine (6.28 mL, 36.1 mmol) was added slowly,dropwise and the reaction mixture was stirred in the ice bath for 5 min,then warmed to room temperature and stirred for 30 min.H-Lys(Z)—OtBu.HCl (103) (7.01 g, 18.8 mmol) was added portionwise,followed by dropwise addition of diisopropylethylamine (6.54 mL, 37.5mmol), and stirred at room temperature for 1 hr. The reaction mixturewas concentrated under reduced pressure, then purified by silica gelchromatography in 10-100% ethyl acetate/petroleum ether to yield 104(8.76 g, 86%, ESI m/z=622.54 [M+H]⁺).

Example. Compound 105

104 (8.76 g, 14.1 mmol) was dissolved in anhydrous methanol (100 mL) andadded slowly along the walls of the 250 mL round-bottom flask containingpalladium on carbon, 10 wt. % (100 mg). A balloon containing hydrogengas was attached to the flask using a three-way stopcock adapter, andthe atmosphere of the flask was evacuated under reduced pressure, thenreplaced with hydrogen gas (3×), then stirred at room temperature underhydrogen gas for 1 hr. To the reaction mixture was added dry, untreatedcelite (˜20 g) and stirred for 5 min. The reaction mixture was filteredand concentrated under reduced pressure to yield 105 (6.86 g,quantitative, ESI m/z=488.46 [M+H]⁺).

Example. Compound 107

Boc-4-aminomethylphenylacetic acid (106) (2.00 g, 7.5 mmol) dissolved ina solution of trifluoroacetic acid (9.75 mL) and triisopropylsilane(0.25 mL) and stirred at room temperature for 30 min, then concentratedunder reduced pressure and coevaporated with dichloromethane (3×), thenplaced under vacuum, to yield 4-aminomethylphenylacetic acid (107)(quantitative).

Example. Compound 108

To a stirring solution of 4-nitrophenyl chloroformate (102) (1.01 g, 5.0mmol) in dry dimethylformamide (10 mL) was added slowly dropwise asolution of 105 (2.45 g, 5.0 mmol) and diisopropylethylamine (0.88 mL,5.0 mmol) in dry dimethylformamide (10 mL), and the reaction mixture wasstirred at room temperature for 30 min under argon. The reaction mixturewas cooled in an ice bath and a suspension of 7 (˜1.25 g, ˜7.5 mmol) anddiisopropylethylamine (1.76 mL, 10.1 mmol) in dry dimethylformamide (10mL) was added slowly dropwise to the reaction vessel, then the reactionmixture was warmed to room temperature and stirred for 30 min underargon. The reaction mixture was purified by preparative HPLC in 10-100%acetonitrile/0.1% formic acid to yield 8 (0.56 g, 16%, ¹H NMR consistentwith structure of 108; ESI m/z=679.50 [M+H]⁺).

Example. Preparation of Protected Ligand 7, Including Coupling Group

Example. Peptide 109

TABLE 1 Reagents for peptide 109 synthesis Molecular weight Reagent mmolEquivalents (g/mol) quantity H-Cys(4- 0.87 1.0 methoxytrityl)-2-chlorotrityl-Resin Fmoc-Asp(OtBu)-OH 2 × 1.74 2 × 2.0 411.5  716 mgPyBOP 2 × 1.73 2 × 2.0 520.39 900 mg diisopropylethylamine 2 × 3.48 2 ×4.0 129.25 606 μL (d = 0.742 g/mL)

In a peptide synthesis vesselH-Cys(4-methoxytrityl)-2-chlorotrityl-resin (0.87 mmol) was loaded andwashed with isopropyl alcohol (3×10 mL) followed by dimethylformamide(3×10 mL). To the vessel was then introduced Fmoc-Asp(OtBu)-OH (2.0equiv) in dimethylformamide, diisopropylethylamine (4.0 equiv), andPyBOP (2.0 equiv). Argon was bubbled for 1 hr, the coupling solution wasdrained, and the resin was washed with dimethylformamide (3×10 mL) andisopropyl alcohol (3×10 mL). Kaiser tests were performed to assessreaction completion. Fmoc deprotection was carried out using 20%piperidine in dimethylformamide (3×10 mL) before each amino acidcoupling. The above sequence was repeated to complete 2 coupling steps.The resin was dried under argon for 30 min.

Example. Peptide 110

TABLE 2 Reagents for peptide 110 synthesis Molecular Reagent mmolEquivalents weight (g/mol) quantity Fmoc-Asp(OtBu)- 0.18 1.0Asp(OtBu)-Cys(Mmt)- 2-ClTrt-resin 108 0.22 1.2 678.81 150 mg PyBOP 0.372.0 520.39 191 mg diisopropylethylamine 0.74 4.0 129.25 128 μL (d =0.742 g/mL)

In a peptide synthesis vessel 109 (0.18 mmol) was loaded and washed withisopropyl alcohol (3×10 mL) followed by dimethylformamide (3×10 mL).Fmoc deprotection was carried out using 20% piperidine indimethylformamide (3×10 mL). Kaiser tests were performed to assessreaction completion. To the vessel was then introduced 108 (1.2 equiv)in dimethylformamide, diisopropylethylamine (4.0 equiv), and PyBOP (2.0equiv). Argon was bubbled for 1 hr, the coupling solution was drained,and the resin was washed with dimethylformamide (3×10 mL) and isopropylalcohol (3×10 mL). Kaiser tests were performed to assess reactioncompletion. Peptide was cleaved from the resin using a cleavage mixtureconsisting of dithiothreitol (114 mg, 0.74 mmol) dissolved in a solutionof trifluoroacetic acid (19 mL). H₂O (0.5 mL), triisopropylsilane (0.5mL). One-third of the cleavage mixture was introduced and argon wasbubbled for 30 min. The cleavage mixture was drained into a clean flask.The resin was bubbled 2 more times with more cleavage mixture, for 30min each, and drained into a clean flask. The drained cleavage mixturewas then concentrated and purified by preparative HPLC in 0-30%acetonitrile/0.1% formic acid to yield 110 (66.9 mg, 43%, ¹H NMRconsistent with structure of 110; ESI m/z=844.57 [M+H]⁺).

Example

Similarly, the following compounds are prepared as described herein:

Example. EC1169 (Compound 12)

In a 25 mL mound bottom flask, 16 (47 mg, 0.04 mmol) was dissolved indimethylsulfoxide (2 mL). A solution of 110 (36 mg, 0.04 mmol) in 20 mMpH7 sodium phosphate buffer (2 mL) was added dropwise, stirring at roomtemperature with Argon bubbling for 30 min. The reaction mixture waspurified by preparative HPLC (10-100% acetonitrile/50 mM NH₄HCO₃ pH7) toyield 112 (56.6 mg, 74%, ¹H NMR consistent with structure of EC1169; ESIm/z=895.58 [M+2H]²⁺).

Example. Synthesis of 3-nitro-2-disulfenylethanol 2

A three-necked 500 mL flask was dried and argon purged, then fitted withan addition funnel. 3-Nitro-2-sulfenyl chloride pyridine 1 (5.44 g,27.11 mmol, 1.4 equiv) was added to the flask and dissolved in 200 mL ofCH₂Cl₂. The solution was cooled to 0° C. Mercaptoethanol (1.33 mL, 18.98mmol) was diluted with 50 mL of CH₂Cl₂ and placed in the additionfunnel. The 2-mercaptoethanol solution was then added drop-wise slowlyover the course of 15 minutes. The reaction progress was monitored byTLC (Rf 0.4 in 5% CH₃OH/CH₂Cl₂). Solvent was removed under reducedpressure and dried. The crude product was purified over silica gel (5%CH₃OH/CH₂Cl₂). The fractions were collected and solvent was removed byevaporating on a rotary evaporator and dried. 3.4 g of3-nitro-2-disulfenylethanol 2 was obtained (77% yield).

Example. Synthesis of4-nitrophenyl-(3′-nitropyridin-2′-yl)disulfenylethyl Carbonate 3

A 250 mL Round-Bottomed Flask was dried and argon purged.3-Nitro-2-disulfenylethanol 2 (3.413 g, 14.69 mmol) was added anddissolved in 45 mL of CH₂Cl₂. 4-Nitrophenylchloroformate (3.663 g, 17.63mmol, 1.2 equiv) was added, along with triethylamine (2.9 mL, 20.57mmol, 1.4 equiv), and the mixture stirred under argon overnight. Themixture was concentrated under reduced pressure and dried. The residuewas purified by silica (30% EtOAc/petroleum ether) and the fractionswere collected, solvent was removed under reduced pressure, and dried.2.7 g of 4-nitrophenyl-(3′-nitropyridin-2′-yl)disulfenylethyl carbonate3 was obtained (47% yield).

Example. Synthesis of 2-(Boc-tubutyrosine (Tut))hydrazinecarboxylic Acid(3′nitropyridyl-2′-yl)disulfanylethyl Ester 6

10.67 g (33 mmol) of Boc-Tut-acid 4 was dissolved in 100 mL anhydrousTHF, 17.24 g (33 mmol) of PyBop, and 17.50 mL (99 mmol, 3.0 equiv) ofDIPEA were added. The reaction mixture stirred for few minutes, 1.0 mL(31.68 mmol, 0.96 equiv) of hydrazine was added and stirred for 15minutes. LC-MS analysis (X-Bridge shield RP18, 3.5 □m column; gradient10% to 100% acetonitrile in 6 min, pH 7.4 buffer) confirmed thehydrazide 5 formation. 14.47 g (36.3 mmol, 1.1 equiv) of4-nitrophenyl-(3′-nitropyridin-2′-yl)disulfenylethyl carbonate 2 wasadded. The resulting clear solution was stirred at room temperature for24 hours. LC-MS analysis (X-Bridge shield RP18, 3.5□m column; gradient30% to 100% acetonitrile in 9 min, pH 7.4 buffer) indicated >98%conversion. The reaction mixture was diluted with EtOAc (˜1.0 L), washedwith sat. NH₄Cl (400 mL), sat. NaHCO₃ solution (3×300 mL), and brine(300 mL). The organic layer was dried over Na₂SO₄ (100 g), andconcentrated under reduced pressure. The crude product was loaded onto aTeledyne Redisep Gold Silica Column and eluted with MeOH/CH₂Cl₂ (330 gcolumn; 0 to 10% gradient) using a CombiFlash chromatography system. Thefractions were collected and solvent was removed under reduced pressureand dried. 16.10 g of 2-(Boc-Tut)hydrazinecarboxylic acid(3′nitropyridyl-2′-yl)disulfanylethyl ester 6 was obtained (82% yield).

Example. Synthesis of Azido Methylbutyrate Dipeptide 9

Dipeptide 7 (10.83 g, 27.25 mmol) was dissolved in 100 mLdichloromethane and imidazole (2.05 g, 1.1 eq.) was added. The reactionmixture was stirred at room temperature to dissolve all solids andcooled in the ice bath for 10 min. TESCl (4.8 mL, 1.05 eqiv.) was addeddrop-wise at 0° C., stirred under argon, and warmed to room temperatureover 1.5 h. TLC (3:1 hexanes/EtOAc) showed complete conversion. Thereaction was filtered to remove the imidazole HCl salt. 125 mLdichloromethane was added to the filtrate, and the resulting solutionwas extracted with 250 mL brine. The brine layer was extracted with 125mL dichloromethane. The combined organic phase was washed with 250 mLbrine, separated, dried over 45.2 g of Na₂SO₄, and filtered. Theresulting solution was concentrated under reduced pressure,co-evaporated with toluene (2×5 mL) and dried over high-vacuum overnightto give 14.96 g of crude product 8.

The crude product 8 was used without further purification. TES protecteddipeptide was dissolved in 100 mL THF (anhydrous, inhibitor-free),cooled to −45° C., and stirred at −45° C. for 15 minutes before addingKHMDS (0.5 M in toluene, 61 mL, 1.05 equiv.), drop-wise. After theaddition of KHMDS was finished, the reaction was stirred at −45° C. for20 minutes, and chloromethyl butyrate (4.4 mL, 1.1 equiv.) was added.The reaction mixture was stirred at −45° C. for another 20 minutes. Thereaction was quenched with 25 mL MeOH and warmed to room temperature.250 mL EtOAc and 250 mL brine were added to the reaction mixture, andthe organic phase was separated. The solvent was evaporated to reducethe volume of solution. The solution was passed through 76.5 g silica ina 350 mL sintered glass funnel. The silica plug was washed with 500 mLEtOAc/petroleum ether (1:4). The filtrate and the wash were concentratedto oily residue and dried under high vacuum to give 16.5 g product 9 asa light yellow wax.

Example. Synthesis of Tripeptide Methyl Ester 10

Based on 16.5 g of alkylated dipeptide 9 (26.97 mmol), N-methylpipecolinate (MEP) (5.51 g, 1.4 equiv.) and pentafluorophenol (7.63 g,1.5 equiv.) were added to a 300 mL hydrogenation flask. NMP (115 mL) wasthen added, followed by EDC (7.78 g, 1.5 equiv.). The mixture wasstirred at room temperature for overnight. 16.5 g of alkylated dipeptide9 was dissolved in 16.5 mL NMP, transferred the solution into thehydrogenation flask, washed the residual 9 with 8 mL NMP, andtransferred into the hydrogenation flask. Dry 10% Pd/C (1.45, 0.05 eq.)was added. The reaction mixture was vacuumed/back filled with hydrogen 3times, and the flask was shaken under hydrogen (˜35 psi) for 3.5 hours.The reaction mixture was analyzed by HPLC. The reaction mixture wasfiltered through 40 g of celite in a 350 mL sintered glass funnel andwashed with 250 mL of EtOAc. The filtrate and the wash were transferredto a separatory funnel and washed with a 1% NaHCO₃/10% NaCl solution(200 mL×3). The organic layer was isolated and dried over 45.2 g ofNa₂SO₄. The solution was filtered and rotovaped under reduced pressure.A sticky amber residue was obtained and dried under high vacuumovernight to give 19.3 g of crude product. The crude product wasdissolved in 10 mL of dichloromethane, split into two portions, andpurified with a 330 g Teledyne Redisep Silica Gold column. The combinedfractions of two purifications were evaporated and dried under highvacuum to give 7.64 g of 10 as a pale yellow solid (overall yield: 39%over 3 steps from compound 7).

Example. Synthesis of Tripeptide Acid 11

Methyl ester 10 (6.9 g, 9.7 mmol) was dissolved in 1,2-dichloroethane(193 mL) and added to a round bottomed flask, equipped with a stir barand condenser. To this solution was added trimethyltin hydroxide (24.6g, 14 eq.). The mixture was heated at 70° C. for 5 hours. LC-MS analysisindicated that the desired product had been formed and <15% of startingmethyl ester 10 remained. The reaction was cooled in an ice bath for 30minutes. The resulting precipitate was then removed by filtration. Thefiltrate was stored overnight at −20° C. The filtrate was then dividedinto two portions and each was subjected the chromatography procedurewhich follows.

Each portion was concentrated under reduced pressure and then placedunder high vacuum for 30 min. The concentrate was then immediatelydissolved in acetonitrile (95 mL). To this solution was then added anammonium bicarbonate solution (95 mL; 50 mM, pH=7). This solution wasloaded onto a Biotage SNAP C18 reverse phase cartridge (400 g,KP-C18-HS) and eluted with 50 mM ammonium bicarbonate and acetonitrile(1:1 to 100% ACN) using a Biotage chromatography system. Fractions wereanalyzed by LC-MS. Pure fractions were combined and ACN was removedunder reduced pressure. The resulting aqueous suspension was extractedwith EtOAc (3×). The combined organic layers were washed with brine,dried over anhydrous Na₂SO₄, and concentrated under reduced pressure.Purification of the two portions resulted in the recovery of clean 11(4.6 g, 65%).

Example. Synthesis of Acetyl Tripeptide Acid 13

In a round bottomed flask, tripeptide acid 11 (3.9 g, 5.6 mmol) wasdissolved in anhydrous THF (23 mL). To this solution was added 3 HF.TEAcomplex (1.8 mL, 2 eq.). The reaction was stirred at room temperaturefor 1 hour. LC-MS analysis indicated complete conversion to the desireddes-TES product 12. The solvent was removed under reduced pressure andthe residue was placed on the high vacuum for 40 minutes. The resultingresidue was then dissolved in pyridine (26 mL), and acetic anhydride(7.9 mL, 15 eq.) and DMAP (25 mg) were added. The reaction was stirredat room temperature for 1 hour. LC-MS analysis indicated completeconversion to the desired acetyl tripeptide acid 13. To the reactionmixture was then added a 1:1 solution of 1,4-dioxane/water (150 mL). Thereaction was stirred for 1 hour at which point the solvents were removedunder high vacuum rotovap. To the residue was added toluene and thesolvent was removed under vacuum (80 mL, 3×). The resulting crude 13 wasdried under high vacuum overnight. The crude material was then dissolvedin ACN (72 mL). Sodium phosphate buffer (50 mM, pH=7.8, 288 mL) was thenadded, and the pH of the resulting suspension was adjusted to neutralusing saturated sodium bicarbonate solution. This solution was loadedonto a Biotage SNAP C18 reverse phase cartridge (400 g. KP-C18-HS) andeluted with water and acetonitrile (20% ACN to 65% ACN) using a Biotagechromatography system. Fractions were analyzed by LC-MS. Clean fractionswere combined, the ACN was removed, and the aqueous solution was placedon the freeze dryer, resulting in purified acetyl tripeptide 13 (2.5 g,71%).

Example. Synthesis of 2-(tubulysin B)hydrazinecarboxylic Acid(3′nitropyridyl-2′-yl)disulfanylethyl Ester 16

The activated Boc-Tut-fragment 6 (2.63 g, 4.42 mmol, 1.1 equiv) wastreated with TFA/CH₂Cl₂ (42 mL; 1:1) and stirred for 30 minutes. LC-MSanalysis (X-Bridge shield RP18, 3.5 □m column; gradient 10% to 100%acetonitrile in 6 min, pH 7.4 buffer) confirmed the product formation.TFA was removed under reduced pressure, co-evaporated with CH₂Cl₂ (3×30mL) and activated Tut-derivative 14 was dried under high vacuum for 18h. In another flask, the tripeptide acid 13 (2.51 g, 4.02 mmol) wasdissolved in 70 mL CH₂Cl₂ (anhydrous) and 1.48 g (8.04 mmol, 2.0 equiv)of pentafluorophenol in 5 mL of CH₂Cl₂ was added, followed by 8.74 g(20.1 mmol, 5.0 equiv) of DCC-resin. The resulting reaction mixture wasstirred at room temperature for 20 hours. LC-MS analysis (X-Bridgeshield RP18, 3.5 □m column; gradient 10% to 100% acetonitrile in 6 min,pH 7.4 buffer) indicated >99% conversion. The DCC-resin was filteredoff, the CH₂Cl₂ was removed under reduced pressure, and thepentafluorophenol activated product 15 was dried under high vacuum for10 minutes. The residue was dissolved in 16.7 mL DMF, and DIPEA (12.6mL, 72.36 mmol, 18.0 equiv) was added. Tut-fragment trifluoroacetic acidsalt 14 in DMF (8.5 mL) was added slowly over 5 min. The resulting clearsolution was stirred at room temperature for 1 h. LC-MS analysis(X-Bridge shield RP18, 3.5 □m column; gradient 10% to 100% acetonitrilein 6 min. pH 7.4 buffer) confirmed the product formation. The reactionmixture was diluted with EtOAc (700 mL), washed with brine (300 mL,2×100 mL), dried over Na₂SO₄ (75 g), concentrated, and dried for 15hours. The crude product was dissolved in CH₂Cl₂ (25 mL) and loaded ontoa Teledyne Redisep Gold Silica Column and eluted with MeOH/CH₂Cl₂ (330 gcolumn; 0 to 5% gradient) using Combiflash chromatographic system. Thefractions were collected and solvent was removed by evaporating on arotary evaporator and dried. 3.91 g of 2-(tubulysinB)hydrazinecarboxylic acid (3′nitropyridyl-2′-yl)disulfanylethyl ester16 was obtained (89% yield).

Example. Preparation of 2-(tubulysin B)hydrazinecarboxylic Acid(pyrid-2-yl)disulfanylethyl Ester 3

Example

Similarly, the following compounds are prepared as described herein:

Example

Additional tubulysins described herein may be isolated from naturalsources, including but not limited to bacteria and other fermentations.Alternatively, the tubulysins described herein may be prepared accordingto conventional processes, including but not limited to the processesdescribed in PCT International Publication Nos. WO 2009/055562, WO2012/019123, and WO 2013/149185, and co-pending U.S. application Ser.No. 13/841,078, the disclosures of each of which are incorporated hereinby reference in their entirety.

Example. Alternative Preparation of EC1169 (Compound 112)

Example

The following representative example compounds are described to betterillustrate the invention described herein and may be prepared accordingto the synthetic methods described for the above examples, and/or usingconventional processes.

Method Example. PSMA Relative Affinity Assay

LNCaP cells are seeded in 12-well Corning Cell-BIND plates and allowedto form adherent monolayers overnight in RPMI/HIFCS. Spent incubationmedia is replaced with RPMI supplemented with 10% HIFCS and containing astandard PSMA binding ligand, such as 100 nM of 3H-PMPA or a competingcompound, such as EC0652, Re-EC652, or ^(99m)Tc-EC0652, in the absenceand presence of increasing concentrations of test compound, such asunlabeled PMPA, or a compound described herein, such as EC1169 orEC1568, a negative control intermediate lacking a PSMA binding ligandwhich is used as a negative control. Cells are incubated for 1 h at 37°C., and then rinsed three times with 0.5 mL of PBS. Five hundredmicroliters of 1% sodium dodecylsulfate in PBS are added to each well;after 5 min, cell lysates are collected, transferred to individual tubesor to vials containing 5 mL of scintillation cocktail, and then countedfor radioactivity. Cells exposed to only the standard PSMA bindingligand, such as 3H-PMPA, or competing compound, such as ^(99m)Tc-EC0652,in FFRPMI (no competitor) are designated as negative controls, whereascells exposed to the standard PSMA binding ligand, such as 3H-PMPA, plus1 mM unlabeled PMPA or competing compound, such as ^(99m)Tc-EC0652 plusRe-EC0652, serve as positive controls. Disintegrations per minute (DPMs)measured in the latter samples (representing nonspecific binding oflabel) are subtracted from the DPM values from all samples. Relativeaffinities are defined as the inverse molar ratio of compound requiredto displace 50% of the standard PSMA binding ligand, such as ³H-PMPA, orthe competing compound, such as ^(99m)Tc-EC0652, bound to PSMA on LNCaPcells, and the relative affinity of the standard PSMA binding ligand,such as PMPA, or the competing compound, such as Re-EC0652, for PSMA isset to 1.

Method Example. Dose Response Assay Against PSMA+ LNCaP Cells

LNCaP cells are seeded in 24-well Corning Cell-BIND plates and allowedto form nearly confluent monolayers overnight in RPMI/HIFCS. Thirtyminutes prior to the addition of test compound, such as a compounddescribed herein, spent medium is aspirated from all wells and replacedwith fresh RPMI. Following one rinse with 1 mL of fresh RPMI/HIFCS, eachwell receives 1 mL of media containing increasing concentrations of testcompound (four wells per sample). Test compound treated cells are pulsedfor 2 h at 37° C., rinsed four times with 0.5 mL of media, and thenchased in 1 mL of fresh media up to 70 h. Spent media is aspirated fromall wells and replaced with fresh media containing 5 μCi/mL³H-thymidine. Following a further 4 h 37° C. incubation, cells arewashed three times with 0.5 mL of PBS and then treated with 0.5 mL ofice-cold 5% trichloroacetic acid per well. After 15 min, thetrichloroacetic acid is aspirated and the cells are solubilized by theaddition of 0.5 mL of 0.25 N sodium hydroxide for 15 min. Four hundredand fifty microliters of each solubilized sample is transferred toscintillation vials containing 3 mL of Ecolume scintillation cocktailand then counted in a liquid scintillation counter. Final tabulatedresults are expressed as the percentage of ³H-thymidine incorporationrelative to untreated controls.

Method Example. Activity In Vivo Against PSMA+ Expressing TumorImplanted in Mice

Four to seven week-old male nu/nu mice (Harlan Sprague Dawley. Inc.,Indianapolis. Ind.) are maintained on a standard 12 h light-dark cycleand fed ad libitum with rodent diet #2918 (Harlan Teklad, Madison, Wis.)for the duration of the experiment. LNCaP cells are grown in RPMI in 10%HIFCS at 37° C. in a 5% CO₂/95% air-humidified atmosphere, harvested andresuspended on ice in matrigel solution (50% RPMI+50% matrigel highconcentration. BD#354248) to a final concentration of 1×10⁶ cells/50 μL.Cell solution and injection needles (28 gauge) are kept on ice prior toinjection and 50 μL of the cell solution injected in the subcutis of thedorsal medial area. Mice are divided into groups of five, seven, ornine, and freshly prepared test compound solutions are injected throughthe lateral tail vein under sterile conditions in a volume of 200 μL ofphosphate-buffered saline (PBS). Intravenous (i.v.) treatments aretypically initiated when the LNCaP tumors are approximately 100-150 mm³in volume. The mice in the control groups do not receive any treatment.Growth of each s.c. tumor is followed by measuring the tumor three timesper week during treatment and twice per week thereafter, until a volumeof 1500 mm³ is reached. Tumors are measured in two perpendiculardirections using Vernier calipers, and their volumes are calculated as0.5×L×W², where L=measurement of longest axis in mm and W=measurement ofaxis perpendicular to L in mm. As a general measure of gross toxicity,changes in body weights are determined on the same schedule as tumorvolume measurements. Maximum % weight loss on any given day due totreatment is determined for each mouse. Survival of animals is monitoreddaily. Animals that are moribund (or unable to reach food or water) areeuthanized by CO₂ asphyxiation.

Example. Relative Affinity of Compounds Described Herein Compared toPSMA Inhibitors DUPA and PMPA

PMPA is reportedly one of the highest affinity ligands, or the highestaffinity ligand, for PSMA. The data in FIG. 1 and FIG. 2 show thatcompounds described herein exhibit higher affinity for PSMA than doesPMPA.

It was unexpectedly discovered that the ligands described herein have ahigher affinity for PSMA than the reportedly highest affinity ligandPMPA. In addition, it was unexpectedly discovered herein that conjugatesof the ligands described herein had even higher affinity for PSMA.

The binding data for additional illustrative compounds described hereinare shown in the following table

Relative PSMA Binding Affinity Example (fold over PMPA = 1.0) EC1080 6EC1067 30 EC1100 20 EC1167 11 EC1168 17 EC1170 7 EC1069 22 EC1183 9EC1241 1.1 EC1303 7 EC1307 28 EC1308 20 EC1310 10 EC1584 6 EC1568 0(negative control)

Example. Dose Response of Compounds Described Herein Against PSMA+LNCaPCells

Using a standard ³H-thymidine incorporation assay as a measure ofcytotoxicity, the data in FIG. 3 show that EC1169 exhibits doseresponsive cytotoxicity against cells in vitro with an IC₅₀ of 13 nM.The corresponding dose responsive cytotoxicity and IC₅₀ values for (V)EC718. IC₅₀ 17.9 nM; (♦) EC1677, IC₅₀ 20.9 nM; (▴) EC1719, IC₅₀ 37.5 nM;(●) EC1720. IC₅₀ 54.2 nM; (▪) EC1721, IC₅₀ 65.6 nM are shown in FIG. 4

Example

Additional compounds described herein against LNCaP cells (2 h-72 h) asdetermined by ³H-thymidine incorporation cells in vitro are shown in thefollowing table.

% ³H-thymidine Example incorporation EC1069 13 nM EC1268 59.1 EC1385 184EC1386 57 EC1387 24 EC1388 12 EC1437 30 EC1550 22 EC1551 20 EC1452 22EC1584 33 EC1588 42

Example. Activity of Compounds Described Herein Against PSMA+ Tumors inVivo

As shown in FIG. 5 treatment of nude mice bearing PSMA-positive LNCaPhuman xenografts with EC1169 (c), EC1550 (●), and EC1551 (▪), each at 2μmol/kg, TIW, 2 weeks, leads to complete responses in all testedanimals. Each compound was compared against vehicle-treated controls(♦). A complete response is observed when the tumor does not appear tohave any net growth during the treatment period of 14 days (the verticaldotted line indicates the last treatment day). As described herein, itis to be understood that the implants comprise the cancer cells in amatrix (100-150 mm³ total volume). Because the matrix remains during theentire observation period, a decrease in the size of the tumor cannotalways be determined by external measurement. It was also surprisinglyfound that, treatment with compounds described herein leads to cure. Forexample, EC169 leads to cure in 2/7 tested animals. A cure is observedwhen the tumor does not appear to grow during the entire observationperiod of 85 days. The data shown in FIG. 5 are the average of themeasurements for each cohort. Therefore, it is to be understood that theincrease in tumor volume beginning at about day 40-45 representsregrowth in the remaining test animals.

Example. Gross Toxicity of Compounds Described Herein

As shown in FIG. 6, the observed efficacy of EC1169 (c), EC1550 (●), andEC1551 (▪), occurred in the absence of weight loss or major organ tissuedegeneration.

Example. Activity of Compounds Described Herein Against PSMA+ Tumors inVivo

Similarly, as shown in FIG. 7, treatment of nude mice bearingPSMA-positive LNCaP human xenografts with EC1584 (▾) and EC1588 (▴),each at 2 μmol/kg, TIW, 2 weeks, leads to complete responses in alltested animals. Each compound was compared against vehicle-treatedcontrols (●). It was also surprisingly found that treatment with EC1588leads to cure in 3/7 tested animals.

Example. Gross Toxicity of Compounds Described Herein

As shown in FIG. 8, the observed efficacy of EC1584 (▾) and EC1588 (▴)occurred in the absence of weight loss or major organ tissuedegeneration.

Example. Activity of Compounds Described Herein Against PSMA+ TumorsCompared to Conventional Chemotherapeutic Agents

As shown in FIG. 9, treatment of LNCaP-tumor bearing mice with docetaxel(the most active chemotherapeutic agent approved for prostate cancer) at10 mg/kg, BIW, 2 weeks, MTD (▴), was found to produce only modestanti-tumor activity, and showed only 1/4 cures, even when administeredat its MTD. In addition, as shown in FIG. 10, that modest observeddocetaxel efficacy was accompanied by high gross toxicity, as evidencedby severe weight loss (18%). EC1169, administered at 2 μmol/kg, TIW, 2weeks (●), is more active and less toxic than docetaxel against PSMA+LNCaP tumors. FIG. 9 shows that treatment with EC1169 leads to acomplete response in all test animals, and resulted in 2/5 cures. FIG.10 also shows that the higher efficacy displayed by EC1169 was notaccompanied by substantially lower toxicity than docetaxcl, providing asignificantly wider therapeutic window. The efficacy of each compoundwas compared to vehicle-treated control (▪).

Example

The in vivo efficacy of (▪) EC1718; (▴) EC1720; (▾) EC1721; (♦) EC1719;and (◯) EC1677; compared to (●) untreated control is shown in FIG. 11.All compounds were administered at 2 μmol/kg, TIW for 2 weeks, beginningon day 21 post tumor implant (PTI). The dotted line indicates the finaltreatment day. The data indicate that the compounds described herein areefficacious in decreasing tumor growth in vivo compared to untreatedanimals. In addition, (▪) EC1718 lead to 1/7 cures; (▾) EC1721 lead to1/7 cures; (♦) EC1719 lead to 2/7 cures; and (◯) EC1677 lead to 4/7cures, where regrowth of the tumor in those animals was not observedduring the observation period. In addition, the compounds describedherein do not show gross toxicity to the test animals, as shown in FIG.12. Without being bound by theory, it is believed herein that the weightchange observed in FIG. 12 for EC1718 at about day 81 is due to theeffects of the tumor size.

Example. Specificity of Compounds Described Herein

PSMA-negative KB tumors did not appreciably respond to EC1169 therapy,supporting the conclusion that the compounds described herein exhibittarget specificity for PSMA-expressing cells.

Example. Hematological Toxicity

Conjugates described herein demonstrate significantly improvedhematological toxicity. EC1169, EC1584, and EC1588 were administered torats i.v. at 0.33 and 0.51 μmol/kg, twice per week (BIW), for 2 weeks.The hematological toxicity in red blood cells and white blood cells wassignificantly lower than untreated controls.

1.-29. (canceled)
 30. A conjugate having a formulaB-L-(D)_(n) or a pharmaceutically acceptable salt thereof; wherein B isa radical of a PSMA binding ligand having the formula

wherein * is the point of attachment to L; L is a 7 atom linkercomprising an alkylenecarbonyl optionally substituted with one or moresubstituents X¹ selected from the group consisting of alkyl, alkoxy,alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, carboxy, carboxyalkyl, and alkyl carboxylateand; D comprises a radioactive isotope of a metal coordinated to achelating group; and wherein n is
 1. 31. The conjugate of claim 30,wherein the alkylenecarbonyl is substituted with one or moresubstituents X¹ selected from the group consisting of alkyl, alkoxy,alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, carboxy, carboxyalkyl, and alkyl carboxylate.32. The conjugate of claim 31, wherein the alkylenecarbonyl issubstituted with an amino group.
 33. A pharmaceutical compositioncomprising a conjugate of claim 30, or a pharmaceutically acceptablesalt thereof, and at least one pharmaceutically acceptable carrier,excipient, or a combination thereof.